Phenobarbital stimulation of cytochrome P-450 and aminopyrine N-demethylase in hyperplastic liver nodules during LD-ethionine carcinogenesis. Microsomes isolated from hyperplastic liver nodules and hepatomas, induced by DL-ethionine, exhibited a reduced cytochrome P-450 content and aminopyrine N-demethylase activity when compared to the organelles of control and surrounding non-nodular liver. Phenobarbital administration to rats caused an increase of microsomal protein, cytochrome P-450 and aminopyrine N-demethylase in all tissue tested. In the hepatoma the rise of cytochrome P-450 and aminopyrine N-demethylase/g of tissue was very low and it is compensated by a slight increase of microsomal protein. In hyperplastic nodules as well as in control and surrounding livers, cytochrome P-450 and aminopyrine N-demethylase increased more than microsomal protein. However, the phenobarbital-induced stimulation was significantly lower in hyperplastic nodules than in control and surrounding livers.


The lack of effect of fasting and alloxan-diabetes on the rate of fatty acid synthesis by some Morris hepatomas. We have measured fatty acid synthesis (estimated by incorporation of [1-14C] acetate by tissue slices) by 3 transplantable Morris hepatomas of different growth rates--9618A, 7794A and 5123C--in response to fasting and alloxan-diabetes. Fasting did not alter fatty acid synthesis by the 3 hepatomas, bringing to 5 the number of tumors not tested and found similarly insensitive. Diabetes caused reduced fatty acid synthesis in 7794A and 5123C but not by 9618A. Host liver fatty acid synthesis was influenced by the presence of the tumor.


Isolation of a factor with toxohormone properties from mouse tumor cells in culture. A small molecular weight factor with  potent toxohormone activity was recovered from the serum-free culture supernatant of mouse MBQA fibrosarcoma cells. The factor depressed plasma-bound iron levels in mice and inhibited cell-free protein synthesis in microgram doses. Preliminary characterizations suggest that the active material is a small peptide.


Two crops of primary lung tumors in BALB/c mice after a single transplacental exposure to urethane. Transplacental exposure of BALB/c mice to urethane on day 17 of gestation resulted in the appearance of 2 distinct crops of primary lung tumors (adenomas). The first, smaller crop occurred in 6--12% of the offspring by 12 weeks of age. The second crop, seen after 9 months of age, consisted of tumors in 45% of the offspring. This finding indicates a potentially useful model system for studying tumor latency. It also suggests caution in the choice of short endpoints in time carcinogenesis assays based on lung tumors in certain strains of mouse.


The effect of combination chemotherapy on the reticuloendothelial system in man. The effect of adjuvant combination chemotherapy (vincristine, CCNU/MeCCNU, 5-fluorouracil) on the phagocytic and catabolic properties of the reticuloendothelial system (RES) was studied in 4 patients before and during the anticancer treatment. The results indicate a transient impairment of both these functions, more prolonged with regard to phagocytosis. No significant changes were noted in the mobile macrophage system. This suggests that chemotherapy induces a depression especially of the stationary macrophage populations in the liver, spleen and bone-marrow.


Carcinogenicity of N-nitrosoephedrine in rats. N-nitrosoephedrine was administered orally to 32 male Srague--Dawley rats at doses of 120 mg/kg body wt. twice weekly. Of the treated animals, 50% died with preneoplastic and malignant lesions mainly in the liver, lung and forestomach. The median time of death of tumor bearing animals was 522 days after the beginning of the experiment. The observation of hyperkeratosis, papillomas, and 1 squamous cell carcinoma of the forestomach suggests that the compound not only exhibits systemic effects but is probably also a weak local carcinogen.


Epithelial alterations in fetal tracheal explants of Syrian golden hamsters exposed to diethylnitrosamine in utero. The fetal tracheae (13th--15th day of gestation) of Syrian golden hamsters exposed in utero to diethylnitrosamine (DEN: 200--400mg/kg body wt.) were divided into cranial and caudal halves. Both sections were then organ-cultured for 4--6 weeks. The cranial explants developed 1.5 times as many epithelial tracheal lining alterations as the caudal explants. These changes were found 25--42 days after the beginning of explant culture and histologically demonstrated hyperplasia, metaplasia and dysplasia. No alterations occurred in the control explants.


Cell surface antigen expression in cytoplasts and karyoplasts of mouse L cells. Karyoplasts and cytoplasts, obtained by enucleation of mouse L cell sublines, express H-2k and L virion antigens in amounts comparable to intact cells.


Alterations of brain and intestine serotonin levels in hamsters pretreated with dietary aflatoxin. Male Syrian hamsters were fed 30.3 ppm mixed aflatoxins (AFT) for a 45-day period followed by a 123-day recovery period. Tests made at the end of the recovery period indicated a reduction in whole brain and duodenal serotonin and a slight but not statistically significant elevation in brain 5-hydroxyindoleacetic acid. There was a high incidence of liver cell hyperplasia in the aflatoxin fed animals. Following toxin withdrawal, the experimental animals showed significant gains in body weight but they never attained a body weight equal to the controls. Aflatoxin apparently interferes with serotonin production and the return to normal body weight even after withdrawal of the animal from the contaminated diet.


Comparison of the metabolic profiles of benzo[alpha]pyrene obtained from primary cell cultures and subcellular fractions derived from normal and methylcholanthrene-induced rat liver. Hepatocyte primary cell (HPC) cultures derived from either (a) non-induced (normal) or (b) methylcholanthrene (MC)-induced rat liver actively metabolized the carcinogen benzol[alpha]pyrene (BaP) over a 24-h period. In both cases, the BaP metabolites generated were qualitatively similar to those seen in the metabolism of BaP by isolated rat liver microsomal fractions; in addition, unidentified compounds were evident in the chromatographic profile generated by the cultured cells. In cells derived from (a), levels of known metabolites (phenols and diols) increased over the time period studied. On the other hand, in cells derived from (b), levels of diols decreased markedly after 8 h. These results suggest that induction with MC enhances both activation and, to a greater extent, conjugative-detoxification pathways of BaP, so that in cells obtained from (b) the formation of water-soluble metabolites is enhanced and levels of organic soluble metabolites are lower than in cells obtained from (a). Metabolism of BaP in primary cell culture derived from rat liver is thus seen to be similar to in vivo metabolism of the carcinogen, but somewhat in contrast to the in vitro microsomal (subcellular) metabolism of BaP where conjugative-detoxification pathways are virtually inoperative.


Inhibition of azoxymethane-induced intestinal cancer by disulfiram. Sprague--Dawley rats, both intact and colostomized animals, were given 24 weekly injections of azoxymethane. Rats were fed either Rat Purina Chow or the same diet plus 0.25% disulfiram. In the intact animals, disulfiram reduced tumors from an average of 6.3 to 0.95. The number of rats developing tumors was reduced from 100% to 60%. In colostomized animals, the reduction was from an average of 5.0 to 0.13. Marked inhibition occurred even in the defunctionalized colon. The results suggest that disulfiram blocks the metabolism of azoxymethane to methylazoxymethanol, and also that the inhibitor may act systemically.


Tumours of the anal region induced in mice by 1,2-dimethylhydrazine. CBA female mice treated with 1,2-dimethylhydrazine developed a high incidence of benign and malignant tumours in the anal region. Many of these tumours originated from the perianal glands rather than the epidermis.


Tocopherol and local X-ray irradiation of two transplantable rat tumours. The influence of tocopherol in an intramuscular dose of 5 mg/100 g body wt. on the effect of local X-ray irradiation of 2 intramuscularly transplanted tumours in the rat was studied. A significantly enhanced effect of irradiation by tocopherol was found, in contrast to the tumour radioprotecting effect with large doses of tocopherol earlier described in literature.


Liver ornithine decarboxylase during phenobarbital promotion of nitrosamine carcinogenesis. The incidence of liver tumors induced in rats by N-diethylnitrosamine was increased by the feeding of phenobarbital. Liver ornithine decarboxylase activity did not increase in animals receiving phenobarbital in the diet, and the concentration of polyamines in the liver was similarly unchanged. No relationship between the promotion of N-nitrosamine-induced liver tumors by phenobarbital and the ornithine decarboxylase activity of the liver was indicated by these experiments.


Effects of dietary butylated hydroxytoluene and phenobarbital on the activities of ornithine decarboxylase and thymidine kinase in rat liver and lung. Rats were entrained to a 12 h dark/12 h light cycle with food (60% protein +/- 0.5% butylated hydroxytoluene or 0.05% phenobarbital) available only during the first 2 h of the dark period. Under these conditions liver ornithine decarboxylase (ODC) activity in control animals displayed a characteristic diurnal oscillation. In livers of rats fed butylated hydroxytoluene or phenobarbital ODC activity was not increased whereas thymidine kinase (TK) activity was stimulated 4--10 fold at 3 days. In lungs from the same animals ODC and TK activities were unchanged. In rats fed butylated hydroxytoluene for 3 days [3H]thymidine incorporation into DNA was increased in liver but decreased in lung.


DNA repair synthesis in primary cultures of kidneys from BALB/c and C3H mice treated with dimethylnitrosamine. A combined 'in vivo--in vitro' autoradiographic method was employed to examine the DNA repair induced in the kidney by a single dose of dimethylnitrosamine (DMNA). Unscheduled DNA synthesis was found to be dose-dependent in primary kidney cultures of DMNA-treated mice, and practically not detectable in controls. Its amount was positively correlated with the different susceptibility of C3H and BALB/c mice to kidney tumor induction by DMNA. This experimental model appears sensitive and able to provide repeatable results. It may be useful to detect the organotropic activity of a carcinogen toward the kidney.


Dimethylnitrosamine-induced structural damage to DNA results from repair of O6-methylguanine rather than repair of 7-methylguanine. DNA isolated from the livers of rats treated with a non-necrotizing dose of [14C]dimethylnitrosamine was fractionated by chromatography of benzoylated-DEAE-cellulose. The preparations of native DNA and DNA containing single stranded regions were then hydrolysed and amounts of methylated guanine determined. Four hours after dimethylnitrosamine treatment there was no difference in the levels of 7-methylguanine and O6-methylguanine in the 2 DNA fractions. By 24 h, although there was no difference in the amount of 7-methylguanine between the DNA fractions, there was a 10-fold difference in the level of O6-methylguanine. The elimination of O6-methylguanine from the fraction of DNA containing single stranded regions is discussed in terms of differing repair processes initiated by 7-methylguanine and O6-methylguanine.


The use of the D-T diaphorase for the detection of foci of early neoplastic transformation in rat liver. This report demonstrates that activity of the enzymes D-T diaphorase and G-6-P dehydrogenases is greatly increased (6- and 4-fold respectively) in hyperplastic nodules produced in the rat liver by dietary acetylaminofluorene. A histochemical technique based on these observations has been developed that permits the visuatlization of early foci of neoplastic transformation in rat liver. The possible physiological implications of these findings are discussed.


2-acetylaminofluorene-induced unscheduled DNA synthesis in hepatocytes isolated from 3-methylcholanthrene treated rats. The dose-dependent induction of unscheduled DNA synthesis (UDS) by 2-acetylaminofluorene (AAF) and N-OH-2-acetylaminofluorene (N-OH AAF) in primary rat hepatocytes isolated from untreated and 3-methylcholanthrene (3-MC) treated rats was investigated. 3-MC treatment was not necessary for the dose-dependent induction of UDS induced by N-OH AAF, suggesting the presence of enzyme levels adequate for its esterification to an active form in isolated primary hepatocytes. Although all doses of AAF increased the level of [3H]thymidine incorporation above that of the control, a dose-dependent response could not be demonstrated, suggesting inadequate constitutive levels of N-hydroxylating enzymes. Treatment of rats with 3-MC 24 h prior to hepatocyte isolation resulted in a dose-dependent induction of UDS by AAF. 3-MC treatment increased the amount of UDS per cell, as well as the percentage of cells induced to repair their DNA.


Comparative tumor initiating activity of 10-methylbenzo-[a]pyrene, 7,10-dimethylbenzo[a]pyrene and benzo[a]pyrene. The tumor initiating activity on mouse skin of benzo[a]pyrene (BaP), 7,10-dimethylBaP, and 10-methylBaP was determined. Each compound was tested at initiating doses of 50 microgram and 100 microgram with promotion by application 3 times weekly of 2.5 microgram tetradecanoylphorbol acetate. BaP induced tumors in 40% (100 microgram) and 25% (50 microgram) of the animals. No tumors were observed in either group treated with 7,10-dimethylBaP. In the groups treated with 10-methyl-Bap, the incidence of tumor bearing animals was 20% at both doses. These results and the results of previous studies on other methylated BaP derivatives suggest that the mechanism of activation of these compounds is similar to that observed for the parent hydrocarbon and probably involves formation of an angular ring diol-epoxide or epoxide.


Influence of tocopherol on tumour cell oxygenation. Tocopherol, a well known tissue anti-oxidant, given before local X-ray irradiation of 2 transplantable rat tumours was previously found to increase significantly the effect of irradiation. In the present study tocopherol did not influence the development of tumour necroses during growth of a transplantable rat hepatoma. The tumour tissue oxygenation of a transplantable rat sarcoma was neither found to be influenced by tocopherol. This suggests that an enhanced tumour radio-sensitivity by tocopherol is probably not explained by an influence on tumour cell oxygenation.


Tumorigenicity of benzo[alpha]pyrene 4,5-,7,8-,9,10- and 11,12-oxides in newborn mice. Benzo[alpha]pyrene (BP) and its 4,5-,7,8-,9,10 and 11,12-oxides were tested for carcinogenicity by intraperitoneal injection into newborn mice. The mice were treated sequentially with 200, 400 and 800 nmol of hydrocarbon on days 1,8 and 15 of life, and the animals were killed at 24 weeks of age. Mice treated with BP, BP7,8-oxide and BP 11,12-oxide had a 93%, 72% and 20% incidence of pulmonary adenomas, respectively, with an average of 10.0, 2.1 and 0.32 adenomas/mouse, respectively. Eight percent of the control animals had pulmonary adenomas with an average of 0.08 adenomas per mouse. The moderate tumorigenic activity of the relatively unstable BP 7,8-oxide, together with earlier data on the strong carcinogenic activity of BP, 7,8-dihydrodiol, supports the idea that BP 7,8-oxide is a proximate carcinogenic metabolite of BP in the newborn mouse.


Influence of agents which modulate thromboxane A2 synthesis or action on R3230AC mammary carcinoma. The effects of agents which modulate thromboxane A2 synthesis or action, were tested in the R3230AC transplanted mammary tumour. Three different inhibitors of thromboxane A2 synthesis or action (copper, dipyridamole and diazepam) all caused an increase in tumour growth. Colchicine and melatonin, both stimulators of thromboxane A2 synthesis, inhibited the growth of the tumour significantly.


Comparison of bladder carcinogenesis by nitrosomethyldodecylamine in Sprague--Dawley and Fischer rats carrying transplanted bladder tissue. Nitrosomethyldodecylamine was given by gavage in oliver oil solution (25 mg twice/week) to male and female Sprague--Dawley and Fischer rats to the back of each of which had been transplanted a suspension of urinary bladder tissue from the same strain. While there was 100% incidence of transitional cell carcinomas in the host urinary bladders, and few other tumors in the animals, there was a tumor in only 1 transplant in a Sprague--Dawley rat, with a possible tumor in another, and none in the Fischer rat, although these were inbred. In a significant number of cases the transplant survived.


Pancreatic ductulitis in Syrian golden hamsters bearing homologous transplantable pancreatic adenocarcinomas. A highly specific pancreatitis primarily affecting the intralobular and intrainsular ductules has been demonstrated in Syrian golden hamsters bearing homologous, non-syngeneic, transplantable pancreatic adenocarcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP). The ductulitis provides further evidence that induced pancreatic neoplasms originate from ductules.


Carcinogenicity of nickel subsulfide for respiratory tract mucosa. The carcinogenicity of nickel subsulfide, Ni3S2, for respiratory tract epithelium was studied in heterotopic tracheal transplants with doses of 1 and 3 mg Ni3S2 per trachea. Chemical determinations indicated that Ni3S2 persisted in the tracheas for seven to nine months. Ni3S2 showed marked toxicity for mucociliary epithelium, resulting in widespread atrophy and focal epithelial necrosis during the first two months of exposure. The submucosa showed mononuclear infiltration and signs of fibroblastic and capillary proliferation. Tumor studies indicated that Ni3S2 can induce carcinomas in tracheal epithelium. The carcinoma incidence was 10% at 1 mg and approximately 1.5% at 3 mg. The higher dose produced a 67% incidence of fibro- and myosarcomas. The data suggest that, compared to some carcinogenic polycyclic hydrocarbons, Ni3S2 may not be a strong carcinogen for the epithelium of conducting airways. The data are discussed in light of other experimental studies and of epidemiological findings on respiratory tract cancers in nickel workers.


Metabolism of [14C]spermidine and [14C]putrescine in normal volunteers and in cancer patients. The administration of [14C]putrescine or [14C]spermidine (i.v., 100 muCi) to normal volunteers or patients with advanced cancer resulted in alpha-phase half-lives of 40 and 30 sec and beta-phase half-lives of 30 and 60 min, respectively. No significant differences were found between the plasma decay curves of normals and those of cancer patients. Urinary excretion was similar with both groups excreting approximately 45% of [ 14C]putrescine within 24 hr and 60 to 76% of [14C]spermidine within 48 hr. Dowex chromatography indicated that more than 90% of the radiolabel in the urine was in a conjugated form. Plasma conjugation studies of [14C]putrescine and [14C]spermidine in both groups indicated near-total conjugation of the radiolabel within 4 to 5 min of i.v. injection. Since putrescine and spermidine are markers of disease activity, characterization of the conjugates will be important for the development of rapid, specific tests of altered disease activity in response to multimodality therapy.


Structure of normal testis and testicular tumors in cyprinids from Lake Ontario. Two types of testicular tumor were evident in cyprinid fishes collected from Lake Ontario. Both types of tumors appeared to be of Sertoli cell origin. One contained predominantly Sertoli cells with little or no lipid content and germinal cells in only small numbers. The second contained large numbers of spermatogonia together with Sertoli cells that contained large amounts of lipid and smooth endoplasmic reticulum. Small, apparently inactive Leydig cells were present in both types of tumors and also in testes of fish not bearing gonadal tumors.


Enhanced development of mammary tumors in rats following transplacental and neonatal exposure to ethylnitrosourea. Transplacental and neonatal induction of mammary tumors (MT's) with ethylnitrosourea (ENU) was studied in Sprague-Dawley rats. A low transplacental ENU dose (10 mg/kg) did not increase the number of MT's or shorten their latency period. High ENU doses (30 mg/kg neonatal, 60 mg/kg transplacental, or 120 mg/kg transplacental) when corrected for differences in life span caused a significant shortening of the tumor induction period and an overall increase in the tumor incidence. With high ENU doses, the MT's were frequently multiple in the same animal and were more often malignant. Tumors developed mostly in females; only a few were observed in males. It is concluded that with a sufficient dose of the carcinogen in susceptible animals, transplacental and neonatal ENU mammary carcinogenesis takes place. The experiment was originally designed to evaluate ENU-induced neurogenic tumors; the results on MT's were obtained incidentally.


Growth patterns and metastatic behavior of human tumors growing in athymic mice. The growth characteristics and metastatic behavior of human tumors growing in athymic nude mice were studied. Human tumor cell lines HEp-2 (carcinoma or larynx) and SW480 (colon carcinoma) were transplanted into athymic nude mice of BALB/c origin. Tumor cells (1 x 10(6) and 2 x 10(7)) were given either s.c. or i.p. Following s.c. injection tumors developed rapidly to become easily palpable with 2 weeks forming a s.c. tumor focus surrounded by a thick fibrous capsule. Animals with s.c. transplants were little affected by the growing tumor. At the time they were sacrificed at Day 34 (HEp-2) and 62 (SW480), a large part of the tumor was necrotic. Capsular infiltration and invasion of lymphatic vessels and perineural and perivascular lymphatic spaces were observed. Metastases to regional lymph nodes were seen in animals kept alive for up to 6 months. Following i.p. transplantation, tumors spread widely in the peritoneal cavity, invaded intraabdominal organs, and metastasized to mediastinal lymph nodes and lungs. Fifteen of 26 animals (60%) developed metastases. Necrosis of the i.p. growing tumors was minimal. All animals in this group died as a result of tumor growth.


Promotion of azoxymethane-induced colonic neoplasia by resection of the proximal small bowel. Potential enhancement of intestinal neoplasia by compensatory mucosal hyperplasia was tested in rats subjected to 50% proximal small bowel resection (PSBR) 10 days after the last of 16 weekly injections of azoxymethane. Azoxymethane alone increased jejunal contents of RNA and DNA each by 26% at 17 to 18 weeks (p less than 0.01) before there was macroscopic evidence of neoplasia. Three months after PSBR alone, ileal hyperplasia was characterized by increased amounts of RNA (42 to 76%) and DNA (68 to 95%), taller villi, deeper crypts, and luminal dilation (p less than 0.05 to 0.001); however, the colon showed only patchy hyperplasia. When the combined effects of azoxymethane and PSBR were observed 26 to 30 weeks after the first injection, rats with PSBR had an increased number of colonic tumors per animal (2.9 versus 1.6 for controls; p less than 0.02). Despite the intense ileal hyperplasia produced by PSBR, ileal neoplasia did not occur. Enhanced colonic carcinogenesis followed sequential exposure of the mucosa to the carcinogen (azoxymethane) and to the promoting factor (PSBR).


An in vitro quantitative assay for tumor cell invasion. An in vitro quantitative assay of tumor cell invasion is described. The assay measures the rate at which [125I]iododeoxyuridine-labeled tumor cells migrate through the chorioallantoic membrane of developing chicken embryos. Invasion chambers were prepared from amber latex cylinders, chorioallantoic membrane, and thread ligatures. The chambers were placed in glass vials to rest on discs of photographic sponge immersed in tissue culture medium. Labeled tumor cells were added to the chamber, and the vial was incubated at 37 degrees. At various time points individual chambers were monitored to determine the number of viable, labeled cells that had passed through the membrane and were present in the medium, on the glass vial, or in the sponge. The application of the assay may be of use in delineating the mechanisms of tumor cell invasion.


Ornithine as a possible marker of cancer. The nonprotein amino acid ornithine is the major source of polyamines in mammalian physiological systems. Increased urinary polyamine levels have been demonstrated in humans with varied types of cancers. The metabolism of DL-[1-14C]ornithine monohydrochloride in rats with either Walker 256 carcinoma or chemically induced methylcholanthrene tumors was studied. Following the i.p. injection of 3 muCi[14C]ornithine per 100 g body weight, the decarboxylation of ornithine-yielding 14CO2 was monitored by utilizing the vibrating reed electrometer-ionization chamber model of Davidson and Schwabe. Tumor-bearing animals showed significant increases in ornithine metabolism as compared to controls; for Walker 256 the tumor-bearing animal to control ratio rose from 1.16 to 1.78, for methylcholanthrene implants it rose from 1.19 to 1.82, and for methylcholanthrene paintings it rose from 1.00 to 2.20. With tumor regression ornithine levels of metabolism in the tumor-bearing animals returned to base line or nearly base-line levels. These results encourage us in our attempt to develop ornithine as a biological marker of cancer.


Mouse skin carcinogenicity tests of the flame retardants tris(2,3-dibromopropyl)phosphate, tetrakis(hydroxymethyl)phosphonium chloride, and polyvinyl bromide. The flame retardants tris(2,3-dibromopropyl)phosphate, tetrakis(hydroxymethyl)phosphonium chloride, and polyvinyl bromide were tested for carcinogenic activity by skin application 3 times weekly in random-bred female ICR/Ha Swiss mice for 420 to 496 days. Tris(2,3-dibromopropyl)phosphate at two dose levels (30 mg and 10 mg/application) induced benign and malignant tumors of the skin, forestomach, and oral cavity (tongue and gingiva) in a statistically significant number of mice (30/group). A statistically significant incidence of papillary tumors of the lung was observed at both dosages, and the higher dose also resulted in one mouse with a tubular adenoma of the kidney. Tetrakis(hydroxymethyl)phosphonium chloride (2 mg/application, 60 mice) and polyvinyl bromide (0.1 ml latex suspension/application, 30 mice) were inactive. Polyvinyl bromide was also injected s.c. into another group of female ICR/Ha Swiss mice once weekly for 48 weeks, and the mice were observed for a total of 60 weeks. Liposarcomas were induced in 19 of 30 mice, which was ascribed to physical carcinogenesis. Appropriate solvent and no-treatment control groups were included.


Changing transplantation characteristics with serial in vivo passage of C3H/He mammary carcinomas. The transplantation immunogenicity of spontaneous C3H/He mammary carcinomas was studied by means of the surgery-challenge procedure during serial in vivo passages in syngeneic mice. Reciprocal cross-sensitization and challenge tests between late transplant generations and early transplant generations (from liquid N2 storage) of the same tumors showed that factors responsible for transplantation resistance and factors causing stimulated tumor growth were present in the tumors at the same time as independent variables. The immunogenicity and the immunosensitivity of tumors were seen as dependent variables. The relative prominence of the characteristics of immunogenicity and growth stimulation changed with continuous in vivo passages. Transplantation immunogenicity was tumor specific in four of five tumors tested. Growth stimulation was, in each of four combinations tested, not tumor specific.


Desmosome ultrastructure and biological behavior of chemical carcinogen-induced urinary bladder carcinomas. In the quantitative electron microscopic study, we examined the relationship of desmosomes to tumor invasiveness in chemical carcinogen (N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide)-induced urinary bladder transitional cell carcinomas in the Fischer rat. The number of a desmosomes per unit area of plasma membrane was significantly reduced in carcinomas. However, the percentage of cell surface area occupied by desmosomes was greater in carcinomas than in controls. This was accounted for on the basis of increases in desmosomal size, which result from squamous differentiation within the tumors. Areas of transitional cell differentiation and squamous differentiation demonstrated an equal capacity for invasiveness. Desmosomes were abundant in invading nests of tumor cells. These findings cast doubt on the validity of the concept of decreased intercellular adhesion as a prerequisite for tumor invasion, since strong interadhesion is probably a function of the area occupied by the intercellular junctions.


Effects of adriamycin on DNA synthesis in mouse and rat heart. Adriamycin induces an inhibition of DNA synthesis in mouse tissues within one hr after treatment. While the effects are short-lived in liver and small intestine, DNA synthesis in heart remains below control values for up to 7 days. After this period DNA synthesis in hearts of treated mice is elevated and remains above control values for as long as 4 weeks. Both 1-beta-D-arabinofuranosylcytosine and actinomycin D also induce inhibition of cardiac DNA synthesis soon after treatment; the effects of 1-beta-D-arabinofuranosylcytosine are over by the end of 24 hr while the effects of actinomycin D persist for at least 4 days. Actinomycin D treatment also induces an "over-shoot" of DNA synthesis in mouse heart. Adriamycin can induce a loss of prelabeled DNA from heart, although no pathological alterations are immediately obvious. The small intestine, however, shows extensive karyorrhexis. The initial effects on cardiac DNA synthesis occur in adrenalectomized animals, indicating that the effects are not mediated via the adrenal gland. We did find, however, that DNA synthesis in heart was sensitive to the effects of starvation. The results of this study indicate that inhibition of mouse heart DNA synthesis is not specific for Adriamycin and that the effects of Adriamycin in heart following a single treatment are long-lived.


Effects of diet on colon carcinogenesis and the immune system in rats treated with 1,2-dimethylhydrazine. The effects of different types of diets on colon carcinogenesis by 1,2-dimethylhydrazine and on the immune system were studied in W/Fu rats. Six different types of diets were used in two sets of experiments. Rats in each group were fed the respective diets immediately upon weaning. 1,2-Dimethylhydrazine dihydrochloride was administered s.c. at a dosage of 15 mg/kg weekly in two divided doses. The rats were followed by sequential laparotomies for the development of gastrointestinal (GI) tumors until death. Tumors appeared earlier, and the total number of GI tumors, particularly those of the colon, was higher in rats fed diet enriched with fat from animal sources. In these rats the GI tumors metastasized more frequently, and their survival, after appearance of the first GI tumor, was significantly shortened. The diet low in animal fat and enriched with carbohydrate reduced the number of GI tumors and delayed their appearance. Semisynthetic elemental diet accelerated the appearance of colon tumors without increasing the total number of GI tumors over the life span of the animals. Serum cholesterol levels evaluated during carcinogenesis suggest a correlation between serum cholesterol levels and the increased frequency of colonic tumors. The alterations in serum immunoglobulin G levels, lymphocyte counts, and surface immunoglobulin-bearing lymphocytes evaluated at different times during carcinogenesis suggested a biphasic ("M type") immune response. Rats fed low residue diets and/or diets containing fat from animal sources had depressed serum immunoglobulin G levels. However, the pattern of immune response was similar in groups of rats fed different types of diets.


Lethal and cytokinetic effects of anguidine on a human colon cancer cell line. Anguidine is a fungal metabolite with antitumor activity in a murine colon cancer model. Because of disappointing results in clinical trials, we analyzed the lethal and cytokinetic effects of anguidine on cultured human colon cancer cells. The studies revealed a moderate reduction in survival only after prolonged drug exposure. Continuous incubation with anguidine for longer than 48 hr produced a moderate increase in the percentage of S-phase cells and a slight decrease in the proportion of cells in G1/0, by pulse cytophotometry. An immediate reduction in the cumulative labeling index for cells continuously exposed to tritiated thymidine and anguidine and a rapid decrease in the cumulative mitotic index for cells continuously exposed to Colcemid and anguidine indicated a block at the G1 into S and G2 into mitosis transitions. Tumoricidal activity of anguidine in a cultured human colon cancer line is poor and requires prolonged exposure. The kinetic data reflect an almost frozen state of the cell cycle.


Maltose tetrapalmitate, a nontoxic immunopotentiator with antitumor activity. The attempt to synthesize a lipid A-like component (the active portion of lipopolysaccharides, but lacking its endotoxic activity) resulted in the production of fatty acyl sugars of which maltose tetrapalmitate was seen to yield the most promising results. It shows no endotoxic activity and elicits an antitumor response in tumor-transplanted animals as shown by (a) an enhancement of the host's capacity to reject a large number of tumor cells, (b) retardation of growth in tumor size, and (c) induction of hemorrhagic necrosis in certain tumors. Experiments with mammary ascites carcinoma show maltose tetrapalmitate to be as effective as is bacterial glycolipid mR595 in its antitumor activity. The degree of sensitivity to maltose tetrapalmitate varies with the tumor-host system: mammary ascites carcinoma less than NH = Cl2TSV5S = B16 less than L26. The mode of action of maltose tetrapalmitate appears to be via its modulation of the immune system. It is itself noncytotoxic to tumor cells in vitro. It is seen to stimulate the spleen cells of certain animals mitogenically, although it causes tumor rejection in all the types of animals tested. Also, it activates peritoneal exudate macrophages in tumor-bearing animals; whether specifically or nonspecifically has not yet been established.


Assay of retinoids in biological samples by reverse-phase high-pressure liquid chromatography. A separation procedure for retinoids based on reversephase high-pressure liquid chromatography with solvent mixtures of acetonitrile and water is described. The method may be applied to the screening of synthetic retinoids, which have potential for use in the prevention of cancer. It is easily adapted to a variety of biological samples and can be applied to other conventional retinoid assays in liver and plasma, detecting as little as 1 nmol retinyl esters and less than 0.3 nmol retinol per g tissue. The one-step chromatography results in separation and simultaneous determination of many of the synthetic retinoids and all of the natural retinoids, including the retinyl esters that are separated into their major fatty acid components. The method has been applied to the analysis of retinoid levels in the liver and intestine of vitamin A-deficient hamsters following a p.o. dose (0.5 mg/day for 2 days) of retinyl acetate or of a synthetic vitamin A analog and is predictive of the degree to which various synthetic retinoids can be converted to retinol and stored in the liver as retinyl esters. Because of its speed, excellent recoveries, and high resolution, the method offers significant advantages over previous, more lengthy procedures.


Heterogeneity and variability of artificial lung colony-forming ability among clones from mouse fibrosarcoma. Lung colony-forming ability of clones isolated from a fibrosarcoma was examined by i.v. injection of the cells cultured in vitro. The clones were heterogeneous in their lung colony-forming ability, demonstrating variability of this malignant characteristic.


Establishment and characterization of three new continuous cell lines derived from human breast carcinomas. Three continuous lines of mammary tumor cells (ZR-75-1, ZR-75-27, and ZR-75-30) have been established from malignant effusions of two women with breast cancer. Differentiated properties expressed by each cell line include: (a) epithelial morphology (by light and electron microscopy) resembling that of the parental tumors; (b) presence of receptors for estrogen and other steroid hormones; and (c) growth responsiveness to estrogen and/or progesterone. All three cell lines possess human karyotypes that differ from one another in modal chromosome number as well as in characteristic marker chromosomes. Two of the cultures (ZR-75-27 and ZR-75-30), although derived from the same patient, have stable differences in their karyotypes.


Immunosuppressive activity and tissue polypeptide antigen content of human ascitic fluids. Ascitic fluids from patients with cancer, cirrhosis, and congestive heart failure and from a patient with noninfectious tuberculosis contain measurable levels of tissue polypeptide antigen (TPA). Only the cancer patients had levels higher than 2.0 microgram TPA per ml. The average TPA levels of 29 cancer patients was 6.4 microgram/ml compared to 0.9 microgram/ml for the controls. Seventeen of 22 cancer ascitic fluids and 7 of 9 fluids from patients with liver disease were immunosuppressive as measured by the inhibition of [3H]thymidine incorporation into phytohemagglutinin-stimulated lymphocytes. Fluids from a patient with congestive heart failure and a patient with noninfectious tuberculosis were not suppressive. We were unable to obtain a significant correlation coefficient between immunosuppression and TPA levels in these fluids. In addition, TPA levels remained constant over a period of 18 months of testing, whereas the in vitro immunosuppressive activity was lost in 9 to 10 months. Sephadex G-200 fractionation of the ascitic fluid resulted in the TPA and immunosuppressive activity eluting in the first large molecular weight peak from the column. Although the 2 activities eluted together in this fractionation, the data suggest that TPA is not responsible for the immunosuppression.


Estradiol and progesterone receptor levels in human breast adenocarcinoma in relation to plasma estrogen and progesterone levels. Plasma estrogen and progesterone levels were determined in 77 premenopausal and 137 menopausal women at the same time that estradiol receptor (ER) and progesterone receptor (PGR) assays were carried out on their breast cancers. The frequency of ER and PGR is approximately the same in premenopausal and postmenopausal women, but the ER content is much higher in postmenopausal women. Although this is usually ascribed to the occupancy of receptors by endogenous estrogen in premenopausal women, our observations suggest that this is unlikely. The higher ER content in postmenopausal women is probably due to the fact that the cyclic progesterone increase in premenopausal women limits estrogen stimulation of ER synthesis. Our data suggest that the circulating levels of estrogen in postmenopausal women are sufficient to stimulate ER and PGR when ER is functional. In premenopausal women, on the other hand, high levels of circulating progesterone may inhibit PGR, and the absence of PGR in the breast cancers of premenopausal women should be interpreted warily if the plasma level of progesterone is unknown.


Plasma protein production by human tumors xenotransplanted in nude mice. For detection of plasma proteins produced by human malignant tumors, a survey of blood plasma obtained from nude mice bearing serially transplanted human tumors was performed by immunoelectrophoresis and the double immunodiffusion technique. Among 34 lines including 18 types of human tumors, human specific plasma proteins were demonstrated in the plasma of nude mice transplanted with two lines of renal cell carcinoma, one adenocarcinoma of the colon, and one squamous cell carcinoma of the maxillary sinus. These tumors can be designated as "ectopic" plasma protein-producing tumors since the organs or tissues from which they originated are not considered to be usual sites of plasma protein synthesis. Plasma protein production, as well as that of alpha1-fetoprotein, was also found in one line of hepatoblasotma and three lines of yolk sac tumors. The above tumors were shown to produce one or more of the following 10 of 20 plasma proteins examined: albumin, prealbumin, alpha1-antitrypsin, ceruloplasmin, alpha2-macroglobulin, hemopexin, haptoglobin, C3 and C4 component of complement, and transferrin. An immunochemical demonstration of human specific cancer products observed in human tumors xenotransplanted into nude mice may provide a new approach for investigating the metabolism of neoplastic cells.


Pharmacokinetics of fluorouracil in humans. A high-pressure liquid chromatography method has been used to investigate the pharmacokinetics of fluorouracil after single i.v. doses to patients. The method is simple and is specific for fluorouracil. No interference was observed in fluorouracil determinations due to other administered medication. Plasma levels of fluorouracil declined rapidly after dosing. The mean half-life was 11.4 min, and drug was essentially cleared from plasma in 1 hr. Individual differences in plasma fluorouracil levels, and also in derived pharmacokinetic constants, were considerably less than those reported previously.


Binding of dibenzo(a,e)fluoranthene, a carcinogenic, polycyclic hydrocarbon without K-region, to nucleic acids in a subcellular microsomal system. Dibenzo(a,e)fluoranthene (DBF), a highly carcinogenic polycyclic hydrocarbon without an apparent K-region, binds covalently to DNA, transfer RNA, and polyribonucleotides when incubated with hepatic microsomal fractions under standard conditions. Optimal binding conditions for [3H]DBF were established. Methylcholanthrene-pretreated mouse liver microsomes induced a higher level of binding of [3H]DBF to DNA than did similarly induced rat liver microsomes. 7,8-Benzoflavone strongly inhibited the binding of this polycyclic aromatic hydrocarbon to DNA, while cyclohexene oxide and trichloropropene oxide had an enhancing effect when used in the presence of rat liver microsomes. An unexpected inhibitory effect was observed with cyclohexene oxide in mouse liver microsome-enriched medium. [3H]DBF bound twice as much to denatured as to native DNA. Incubation of [3H]DBF in the presence of liver microsomes and polyribonucleotides (polyadenylate, polyuridylate, polyguanylate, and polyinosinate) indicated that binding occurs mainly with guanine. Binding of [3H]DBF to DNA of various origins was found to be directly proportional to the amount of GC pairs. Preliminary results indicate a covalent bond between DBF and nucleic acids.


Increase of urinary putrescine in 3,4-benzopyrene carcinogenesis and its inhibition by putrescine. A significant increase in putrescine was noted in the urine of mice with experimental s.c. tumors induced by a single injection of 3,4-benzopyrene solution (2.52 mg of 3,4-benzopyrene in 0.5 ml of tricaprylin). When 10 mg of putrescine were added to the 3,4-benzopyrene solution, the development of tumors was completely inhibited and the increase of urinary putrescine in mice was suppressed simultaneously. Animal weight data of a control group receiving only putrescine indicated that the inhibitory effect of putrescine is not due to its toxicity.


Inhibition of nucleoside uptake into Ehrlich ascites carcinoma cells by new cytostatic methylhydrazones. For the purpose of studying the structure-activity relationships of N'-methyl-N'-beta-chloroethylbenzaldehyde hydrazones, new hydrazones were synthesized in which the beta-chloroethyl group was replaced by substituents conveying a partial positive charge at the N' moiety by induction and/or mesomerism. In a preliminary antitumor evaluation, some of these hydrazones showed a cytostatic activity in vivo similar to that of the beta-chloroethyl hydrazones. The new hydrazones also strongly inhibit the uptake of nucleosides into tumor cells and intensify the in vivo cytostatic effect of methotrexate.


Effect of hyperthermia on the immune response of normal rabbits. Sequential skin responses to dinitrochlorobenzene challenge and repeat assays of serum antibody titer after two injections of bovine serum albumin were used as functional indices of cellular and humoral immunocompetence following hyperthermia in normal adult New Zealand White rabbits. The animals were subjected to different degrees of local hyperthermia by watercuff or radio-frequency heating of the normal thigh muscles maintained at 42 degrees for 1 hr on 3 consecutive days or 47--50 degrees for 30 min, respectively, or to total body hyperthermia (42 degrees for 1 hr on three occasions) in a humidified incubator. No alteration occurred in the response of heated rabbits to dinitrochlorobenzene challenge over a 3-month period. The humoral immune response to bovine serum albumin was significantly depressed (p less than 0.02) in the treated animals, and the reduction was independent of method and degree of heating. The results suggest that the B-lymphocytes are more susceptible to hyperthermic damage than is the T-cell population.


Presence of RNA in the nucleoprotein complex spontaneously released by human lymphocytes and frog auricles in culture. Cell systems as different as normal human blood lymphocytes and frog auricles release spontaneously a nucleoprotein complex in their culture medium. This release seems to be an active mechanism that is unrelated to cell death. The presence of RNA in this complex is demonstrated. The amount of extracellular RNA is regulated by the same homeostatic mechanism that has previously been shown to govern DNA release in the same cell systems. This extracellular RNA is linked by hydrogen bonds to the extracellular DNA and cannot be extracted by a usual phenol procedure, due perhaps to the presence of a glycoprotein. Further purifications by chloroform, sodium perchlorate, and hydroxyapatite are necessary to obtain an RNA molecule that is acid precipitable, RNase and KOH sensitive, and orcinol positive. The extracellular RNA sediments between 2.5 and 4S and is not a transfer RNA. It is more highly methylated than the 28S, 18S, and 4 to 5S cellular RNA. It activates DNA synthesis in vitro.


Effect of 5-fluorouracil and thymidine on the transmembrane potential and zeta potential of HeLa cells. The main result of the experiments reported in this paper is the observation that HeLa cells, when treated with 5-fluorouracil (5-FU), show a damped oscillatory response in the transmembrane potential. This effect is counteracted by thymidine. Damped oscillation in the transmembrane potential occurs within the first hr after addition of 5-FU, commencing with an initial hypopolarization. At low concentrations (10(-8) through 10(-7) M), hyperpolarization of the cell membrane is observed after lengthy incubation time (24 to 96 hr). Membrane depolarization is enhanced at higher concentrations (10(-6) through 10(-4) M) and with increasing exposure to the cytostatic substance. The surface charge or zeta potential of HeLa cells is altered to a lesser extent by 5-FU than is the transmembrane potential. Distinct effects can be observed as late as after 72 and 96 hr. When thymidine (2 x 10(-5) M) is added along with 5-FU, the depolarizing action of the antimetabolite can be abolished. Thymidine itself, at the concentration used, causes almost no change in membrane polarization. The results suggest that the effect of 5-FU on the cell membrane is related to the influence of the antimetabolite on cell growth. Moreover, this membrane effect might be the primary cause for the cytostatic action.


Establishment of epithelial cell lines following exposure of cultured tracheal epithelium to 12-O-tetradecanoyl-phorbol-13-acetate. In vitro exposure of rat tracheal epithelium to the tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate results in a marked increase in growth capacity. The growth changes are manifested in an increased rate of cell division and growth in primary cultures and in the establishment of permanent epithelial cell lines. Such changes did not occur in control cultures. Fourteen of the cell lines have been inoculated into immunosuppressed recipients, and all are nontumorigenic.


Adriamycin and mitomycin C: possible synergistic cardiotoxicity. Ninety-one patients with advanced breast cancer failing adriamycin (ADR)-containing combination chemotherapy were treated with a combination of mitomycin C (MMC) and megestrol acetate. Congestive heart failure (CHF) occurred in 14 (15.3%) of 91 MMC-treated patients compared to three (3.4%) of 89 patients treated with similar ADR-containing combination chemotherapy without MMC (P = 0.01). The median time from the last dose of ADR to evidence of CHF was 8.5 months for the MMC group compared to 1.5 months for the other group. A significantly higher incidence of late onset of CHF implicates MMC as a possible cardiotoxic agent.


Combination chemotherapy for ovarian carcinoma with cyclophosphamide, adriamycin, and cis-dichlorodiammineplatinum(II) after failure of initial chemotherapy. Twenty-four patients who had stage III or IV ovarian adenocarcinoma and had failed prior chemotherapy were treated with cyclophosphamide, adriamycin, and cis-dichlorodiammineplatinum(II) every 3 weeks. Twelve patients had objective responses and an additional six had subjective improvement. The median duration of survival for responders is 9 months. There were no instances of nephrotoxicity. Fluid management consisted of 1000-2000 ml of iv fluid without Lasix- or mannitol-induced diuresis. Patients responded after having failed radiotherapy or multiple-drug chemotherapy, including some who were 70-75 years of age or were greater than 50% bedridden. Patients with one- or two-drug prior chemotherapy had a higher response rate (ten responses among 17 patients) than those previously treated with three or more drugs (two responses among seven patients).


Sample size requirements for evaluating a conservative therapy. Determination of an adequate sample size for a clinical trial has traditionally involved the specification of type I (false positive) and type II (false negative) error rates, and a difference that one wishes to detect. Because newer therapy has generally been more invasive or more toxic, it is conventional for the type I error to be 0.05 in order that new therapy not be accepted as superior unless its advantages are definitively established. Recently, many new trials have been directed toward showing that a more conservative treatment is equivalent in efficacy to a standard intensive therapy. In this paper, we provide formulas which prescribe the sample size necessary to meet certain criteria specified by the investigator for this alternative type of clinical trial. In addition, the percent increase in total sample size is described when more patients are allocated to one treatment than the other.


Antitumor properties of a new folate analog, 10-deaza-aminopterin, in mice. A new folate analog, 10-deaza-aminopterin, was substantially more active than methotrexate, following sc administration in mice, against three of five ascites tumors and two of three solid tumors. For ascites tumors, maximum increases in lifespan (using 6--12 mg/kg q2d X 5) with 10-deaza-aminopterin versus methotrexate were + 171.2%/+ 149.8% against L1210 leukemia, +118.4%/+109.1% against P815 plasmacytoma, +64%/+20.9% against Ehrlich ascites carcinoma, +84.2%/+44.8% against Taper liver tumor, and greater than +159.6%/+64.0% against Sarcoma 180 with longterm survivors after 10-deaza-aminopterin. In a smaller number of experiments comparing LD10 dosages (given q2d X 5) of aminopterin, methotrexate, and 10-deaza-aminopterin, aminopterin was the least effective and 10-deaza-aminopterin was the most effective against the L1210 leukemia, Sarcoma 180, and Ehrlich ascites tumors. Following oral administration (3--6 mg/kg q2d X 5), a twofold greater increase in survival time was obtained against the L1210 leukemia with 10-deazaaminopterin (+122.8%) versus methotrexate (+57%). At a dosage of 6 mg/kg q1d X 5 against solid tumors, the relative tumor volumes (treated/control X 100%) were 12%/41% for Sarcoma 180, 16%/31% for Taper liver tumor, and 20%/30% for Ehrlich ascites carcinoma. Overall, the data suggest a broader spectrum of effective antitumor action in mice and a potential for the expanded clinical utility of this category of agent.


Effects of 5-fluorouracil on human colon carcinoma and solid rat Walker 256 carcinosarcoma: evaluation as in vitro predictors of clinical response. Several biochemical effects of 5-fluorouracil (5-FU) including inhibition of the incorporation of 3H-deoxyuridine (3H-UdR) into DNA, inhibition of ribosome formation, and formation of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) were examined in samples of human colon carcinomas to determine if any of these drug effects might have predictive value as a reliable guide to 5-FU therapy. For comparison, the solid rat Walker 256 carcinosarcoma, which is only minimally responsive to 5-FU, was also studied. For each of the biochemical effects of 5-FU measured in the various samples of Walker 256 tumors, the responses were consistent and varied within a narrow range. In contrast, the formation of FdUMP from 5-FU and the degree of inhibition of the incorporation of 3H-UdR into DNA by 5-FU were extremely variable in the population of human colon carcinomas examined. In all human tumors examined, 5-FU caused a reduction in the formation of ribosomes, but even in the absence of 5-FU, the total amount of ribosome synthesis was so low that it makes measurement difficult to quantitate. Based on our data, a study might be warranted to determine if there is a correlation between FdUMP formation and responsiveness of colon carcinoma to 5-FU therapy.


Glycerol: a successful alternative to dexamethasone for patients receiving brain irradiation for metastatic disease. Thirty-three patients with metastatic brain neoplasms of various types received glycerol instead of corticosteroids during periods of brain irradiation. In the 25 symptomatic patients, responses from this treatment were seen in those patients whose primary symptom was vomiting (ten of 12 patients), headache (nine of ten), papilledema (five of nine), paralysis (six of eight), confusion (six of seven), and dysphasia (four of six). Glycerol was well tolerated; it did not induce immunosuppression when administered in combination with radiotherapy and chemoimmunotherapy. Further investigation is indicated to compare its efficacy with that of dexamethasone.


Lack of therapeutic synergism between vincristine and methotrexate in L1210 murine leukemia in vivo. In vivo studies were performed in mice bearing L1210 ascites tumor to examine the interaction of vincristine (VCR) at doses of 0.15 and 1.5 mg/kg with methotrexate (MTX) at doses of 0.5, 5, 50, and 350 mg/kg. The combination was administered on Days 2, 6, and 10 after tumor inoculation. VCR was given either concurrently with MTX or preceding it by 30, 60, or 120 minutes. VCR administered at a dose of 0.15 mg/kg achieved a peak peritoneal fluid concentration of 1.4 micron declining with a half-life (T1/2) of 33.5 minutes and produced no in vivo augmentation of MTX uptake. VCR administered at a dose of 1.5 mg/kg achieved a peak peritoneal fluid concentration of 16.5 micron declining with a T1/2 of 24.9 minutes and produced significant in vivo augmentation of MTX uptake when given concurrently with the MTX and 30 minutes prior to MTX at a dose of 350 mg/kg. No other interval was associated with augmented MTX uptake. When animal survival was evaluated, no therapeutic synergism was observed at any dose level of either drug in any schedule. In fact, VCR (0.15 and 1.5 mg/kg) and MTX at doses of greater than or equal to 50 mg/kg produced toxic synergism which adversely affected survival. Since the drug doses studied are comparable with those used in "high-dose" clinical protocols, individual tumor evaluation is indicated to support the use of these drugs in combination.


Prediction of adriamycin disposition in cancer patients using a physiologic, pharmacokinetic model. A ten-compartment flow-limited pharmacokinetic model scaled from rabbit tissue distribution data was used to predict plasma adriamycin concentrations in 23 patients and adriamycin tissue uptake in nine surgery patients following iv bolus doses of 10--60 mg/m2. The predicted concentrations were compared to experimentally determined adriamycin using a specific thin-layer chromatographic fluorescence scanning procedure. The predicted plasma time course for 11 of 16 patients with relatively normal liver and kidney function agreed closely with the observed plasma time course. Deviations in the other five patients were ascribed to possible changes in the profile of metabolite formation and/or fluctuations in biliary clearance. All four patients with elevated serum bilirubin demonstrated significantly  higher and more prolonged plasma levels than predicted. The results of two patients with impaired kidney function and one patient with both hepatic and renal involvement were inconclusive. The comparison between predicted and observed tissue concentrations in biopsy samples was varied; however, all were within an order of magnitude. It is concluded that the model depicts adriamycin uptake and distribution reasonably well; however, more needs to be known concerning individual variation in metabolic and biliary excretion rates for this to become more patient-specific. Also, a tumor compartment appears to be an important addition in modifying the model to allow for clinical utility.


5-year survey of methotrexate, cyclophosphamide (Endoxan), and vincristine (MEV) therapy for advanced non-Hodgkin's lymphomas. Five courses of a combination chemotherapy regimen with methotrexate, cyclophosphamide (Endoxan), and vincristine were given to 64 patients, 37 with lymphocytic lymphoma (LL) and 27 with histocytic lymphoma (HL). Eighteen of the 37 patients with LL (48%) achieved a complete remission (CR); 12 of these 18 patients have been in CR for 20--58 months. Among the complete responders only two died. Twenty-two of the 27 patients with HL (81%) achieved CR and 12 of these patients are still in CR. Ten of the complete responders have been relapse-free for 24--70 months and five have been relapse-free for greater than 60 months. Clinical and hematologic tolerance was such that the interval between each course had to be prolonged in only three of the 64 patients and, with the exception of vincristine, the dosages were not reduced.


cis-Dichlorodiammineplatinum(II) alone followed by adriamycin plus cyclophosphamide at progression versus cis-dichlorodiammineplatinum(II), adriamycin, and cyclophosphamide in combination for adenocarcinoma of the lung. Forty-one patients with proven metastatic adenocarcinoma of the lung were randomized into two comparable groups after stratification for performance status and presence of measurable or evaluable disease. Treatment 1 consisted of cis-dichlorodiammineplatinum(II) (P) at a dose of 15 mg/m2/day by iv push on Days 1--5 every 4 weeks. Upon progression or the onset of renal toxic effects, patients were crossed over to cyclophosphamide (C) at a dose of 400 mg/m2, and adriamycin (A) at a dose of 40 mg/m2, both given by iv push on Day 1 every 4 weeks. Treatment 2 (CAP-I) consisted of C-A in the same doses as above plus concurrent P, 40 mg/m2 by IV push on Day 1 every 4 weeks. Eight patients receiving P developed serum creatinine values greater than or equal to 1.5 mg/100 ml versus one patient receiving CAP-I (P = 0.05). Objective regressions occurred in two of 22 patients with P, five of 17 with C-A, and eight of 19 with CAP-I (P = 0.025; CAP-I versus P). There was a significant increase in the median duration of regression in patients responding to C-A following P (267 days) versus patients responding to CAP-I (95 days). The improved rate of response with CAP-I and the prolonged duration of response with C-A following P suggest a potentiating effect between P and C-A whether given simultaneously or sequentially.


Steroid-nucleosides.

Steroid-nucleosides. A new class of potential chemotherapeutic agents consisting of steroids coupled with nucleoside bases is described. Such structures may be viewed as nucleosides in which the sugar moiety is replaced by a steroid molecule. We may therefore expect the nucleoside characteristics to give interference at the DNA level, resulting in the desired antitumor activity, whereas the steroid moiety may provide target specificity. A number of such coupling products were prepared and preliminary biologic studies are reported.


Association between steroid hormone receptors and response rate to cytotoxic chemotherapy in metastatic breast cancer. The influence of steroid hormone receptors on response rate to cytotoxic chemotherapy in 70 patients with metastatic breast cancer was determined in a retrospective study. We have previously reported that 34 of 45 patients with tumors containing low or absent estrogen-receptor values had objective responses to chemotherapy while three of 25 patients with positive estrogen-receptor tumors responded. In the present study, 22 of 34 patients with low or absent progesterone-receptor tumors had an objective response to cytotoxic chemotherapy, while none of eight patients with a positive progesterone-receptor tumor responded (P less than 0.05). Patients having tumors with a negative estrogen receptor and a negative progesterone receptor had a response rate of 88% (21 of 24 patients). There were three patients whose tumors were estrogen-receptor negative but progesterone-receptor positive; none had a response to chemotherapy. Chemotherapy response was not associated with the presence or absence of either androgen or glucocorticoid receptor. We conclude that progesterone-receptor values in addition to estrogen-receptor status may prove to be important correlates of response to cytotoxic chemotherapy in metastatic breast cancer. Androgen- and glucocorticoid-receptor analyses are not helpful in predicting response to chemotherapy.


Pharmacokinetics of BCNU in man: a preliminary study of 20 patients. Using the technique of direct sample insertion selected ion monitoring chemical ionization mass spectroscopy, BCNU levels were measured in vivo in patients and in vitro in serum, sera ultrafiltrates, and buffered Ringer's solution. The disappearance of BCNU in vitro was found to be first-order with a half-time of 11.6 minutes (+/- 0.5 SD) in volunteers and 15.6 minutes (+/- 2.3 SD) in patients. The disappearance from serum was catalyzed by a macromolecular component of the serum and was slowed by serum lipids. Analysis of the pharmacokinetics of BCNU in 20 patients using a two-compartment open model demonstrated a volume of distribution of 3.25 liters/kg (+/- 1.69 SD), a clearance of 56 ml/minute/kg (+/- 56 SD), and a transfer constant from the central compartment to the outside (K10) OF 0.0324 MINUTE-1 (+/- 41% SD), which was close to the decomposition rate observed for BCNU in serum in vitro. The pharmacokinetics of BCNU in patients may be affected by the percent of body fat and the lipid content of the serum.


Kinetics of cytotoxicity of VM-26 and VP-16-213 on L1210 leukemia and hematopoietic stem cells. The in vivo effects of VM-26 and VP-16-213 upon hematopoietic stem cells and L1210 leukemia cells were quantitated using the spleen-colony assay technique. The effect of VM-26 on leukemia cells was further quantitated using an endpoint dilution assay. The dose-response curves for leukemic clonogenic cells for both agents when given by iv injection were exponential and biphasic, indicating the existence of two populations. The survival of leukemia cells when VM-26 was administered as a 24-hour infusion was less than that found for equivalent doses administered as single injections. The survival kinetics with respect to time for a low dose (0.02 mg/mouse) and a high dose (0.15 mg/mouse) of VM-26 were similar in that the decrease in survival was rapid, reaching a maximum effect within 4 hours after administration. With the low dose, repopulation of the femoral marrow started immediately, whereas with the high dose, there was a 20-hour delay in repopulation. The data from the increase in lifespan studies were in excellent agreement with the quantitative assays. The dose-response curves for the normal hematopoietic clonogenic cells were also exponential, but these cells were much less sensitive to the agents than were the leukemia cells.


Estramustine phosphate in the treatment of advanced malignant melanoma. Twenty-six patients (14 men and 12 women) with histologically proven advanced malignant melanoma, who previously had not responded to DTIC, methyl-CCNU, and procarbazine, received estramustine phosphate (estracyt) (15 mg/m2) in daily divided doses. All patients had measurable disease. One patient developed a complete remission and one patient had improvement in liver function without measurable regression in the tumor. In three other patients (11%), the disease remained static for a period of 3--5 months. The mean survival time from the beginning of therapy was 16.8 months for the patients with a response or static disease and 2.18 months for those who had no response. Gastrointestinal toxicity was minimal; no hematologic toxicity was observed. It appears that estramustine phosphate used as a single agent for treating advanced malignant melanoma after patients failed to respond to DTIC and to the combination of methyl-CCNU and procarbazine has a poor response rate.


Chemo-hormonal therapy for metastatic renal cell carcinoma with adriamycin, hydroxyurea, vinblastine, and medroxyprogesterone acetate. In a phase II study, eight patients with metastatic renal cell carcinoma have been treated with a combination of chemotherapy and hormonal therapy using adriamycin, hydroxyurea, vinblastine, and medroxyprogesterone acetate. Five patients have responded, including one with complete response, one with subjective improvement, and three with partial responses. The median survival was 4 months with a range of 4--11 months. With the exception of mild alopecia and nausea, toxicity was minimal; no significant hematologic toxicity was noted. The initial results of this study suggest that chemo-hormonal therapy plays a significant role in the management of renal cell carcinoma.


Phase II study of melphalan in colorectal carcinoma. In a prospective phase II study, 25 patients with advanced (Duke's D) colorectal adenocarcinoma received 0.25 mg/kg of melphalan orally daily for 4 days every 28 days. There were 17 men and eight women. All patients had measurable areas of known malignant disease which served as objective indicators of the response to chemotherapy. All patients were evaluated for at least two cycles of therapy. Each patient had had previous treatment with 5-fluorouracil and, in addition, 24 of 25 patients had had treatment with methyl-CCNU; all patients had disease progression with both regimens. Toxicity consisted of mild gastrointestinal symptoms and transient leukopenia (wbc count less than 4000/mm3) in nine of 25 (36%) patients and thrombocytopenia (platelet count less than 100,000/mm3) in six of 25 (24%). One of 25 (4%) patients had an objective response for 12 weeks, 15 of 25 (60%) had disease progression, and nine of 25 (36%) remained stable for 2 months. We conclude that melphalan is ineffective for patients with metastatic colorectal carcinoma who have previously failed to respond to both 5-fluorouracil and methyl-CCNU.


Dissecting the hematopoietic microenvironment. II. The kinetics of the erythron of the S1/S1d mouse and the dual nature of its anemia. An analysis of the S1/S1d mouse for ferrokinetics and fate of peripheral red blood cells has shown the cause of its anemia to be dual in nature. While the S1/S1d produces red cells at a slightly greater rate than its normal littermate, its bone marrow and spleen appear to be operating near their maximal capacity and will reduce their output if anemic stress is partly relieved. The cause of the moderately high level of erythropoiesis in the S1/S1d is a mean daily loss of 2.5--3.0% of its total blood volume via the intestinal tract.


Effect of myleran on murine hemopoiesis. III. Changes in the density distribution of spleen colony forming (CFU-S) and agar gel colony forming cells (CFU-C). Spleen colony forming cells (CFU-S) and agar-gel colony forming cells (CFU-C) are separate but heterogeneous cell populations, as measured by buoyant density. Myleran (MY) abrogated the major (lighter density) components of the CFU-S and CFU-C compartments, thus shifting the surviving CFU-S and CFU-C density profiles into the higher density region. The normal spleen colony erythrocytic : granulocytic (E:G) ratio profile showed three density regions with different distributions of erythroid and granulocytic colonies. The preponderantly erythrocytic colony-generating CFU-S of the intermediate density regions were eradicated by MY. Comparison of the density distribution of erythrocytic and granulocytic colony-generating CFU-S of normal bone marrow showed that the erythrocytic CFU-S profile paralleled that of total CFU-S, while most of the granulocytic CFU-S were contained in the major (and lowest density) peak. MY eradicated the two main (and lowest density) peaks of CFU-S; surviving CFU-S occurred preponderantly in a minor (higher density) peak which has a high potential for generating erythrocytic colonies.


The response of the satellite and other non-neuronal cells to the degeneration of neuroblasts in chick embryo spinal ganglia. The responses of non-neuronal cells to the degeneration of neuroblasts were studied in the spinal ganglia of the chick embryo. Reactive changes of the non-neuronal cells were not apparent during the early stage of degeneration of the neuroblast, but during the later stages of degeneration evidence was found suggesting that non-neuronal cells subdivided and phagocytosed the affected neuroblasts. Three types of non-neuronal cells appeared to participate in the phagocytosis: satellite cells, cells resembling undifferentiated elements, and macrophages. The appearance of the latter coincided with the beginning of the vascularization of the ganglionic rudiment. The phagocytic capacity of satellite cells is discussed in the light of these and other studies by light and electron microscopy.


Gap junctions in human sebaceous glands. An examination of human seabaceous glands by transmission electron microscopy has revealed the presence of gap junctions. The junctions are found in abundance between differentiating cells, and annular forms are also seen. The possible significance of this new finding is briefly discussed.


Neurosecretion. XVII. Experimentally induced release of neurosecretory material by exocytosis in the insect Leucophaea maderae. In the corpora cardiaca of the insect Leucophaea the administration of serotonin elicits ultrastructural features indicative of the extrusion of neurosecretory material by exocytosis. The response to the stimulus and the process of extrusion seem to occur at considerable speed. Nearly all of the 30 test animals, fixed at various intervals starting as early as 3 min after the injection of the drug, show granules captured at the moment of leaving the axon as well as fully exteriorized secretory material. The fact that many of these granules are much smaller than the typical neurosecretory type speaks for intracellular fragmentation of the latter prior to the discharge of this cellular product. After 25 min or more the extruded electron dense structures show signs of breakdown. 3the apparent speed of these phenomena accounts for the dearth of omega-type configurations observed in unstimulated specimens of this species. The possible relationship between the membrane phenomena involved in exocytosis and the transient protrusions of bounding membranes of neurosecretory granules described in earlier papers remains to be clarified.


Effects of vitamin A deficiency on ultimobranchial cysts in the thyroid gland: an electron microscopic study. Ultimobranchial cysts in the thyroid glands of rats receiving a diet adequate in vitamin A are lined with stratified squamous epithelium and contain non-keratinized cellular debris. The epithelium of these cysts in vitamin A deficient animals is keratinized, and their lumina contain keratinized cellular strands surrounding a core of cellular debris. Upon return to a diet adequate in vitamin A the epithelium returns to a non-keratinized state, and the lumina contain keratinized strands surrounded by cell fragments and desquamated whole cells. Occasionally these cysts have an epithelium that is highly irregular in appearance. The relationship of alterations in this tissue to possible subsequent development of neoplasias is discussed.


Ultrastructure and segmentation of microdissected kidney tubules in the marine flounder, Pleuronectes platessa. A new method combining electron microscopy with microdissection was used to study the segmental variation along the tubule of a marine flounder. Two different nephron types were present. One type had long tubules with the glomeruli located close to the kidney surface. The other type had shorter and more coiled tubules with the glomeruli located close to the terminal end of the same nephron. Five different segments were present: (1) neck segment, (2) first proximal segment, (3) second proximal segment, (4) third proximal segment, and (5) collecting tubule. The third proximal segment was not present in all tubules. An extensive system of infoldings from the plasma membrane was present in all segments, except the neck segment and the collecting tubule. Tight junctions impermeable to lanthanum were present in all segments. The collecting duct cells also had extensive infoldings from the plasma membrane and tight junctions impermeable to lanthanum were also present here.


The effects of various stimuli on the cellular composition of peritoneal exudates in the mouse. A comparative study on the composition of cell populations collected from the unstimulated mouse peritoneal cavity and at various intervals after the intraperitoneal injection of glycerol trioleate, casein, paraffin oil, glycogen, sodium chloride, and proteose peptone, was carried out. The cellular composition of the peritoneal exudates, especially with respect to the ratio of monocytes to resident macrophages, was shown to be dependent on the nature of the stimulus and interval after stimulation.


Kinetics of gametogenesis. II. Comparative autoradiographic studies of oogonia and multiplying prospermatogonia of the Wistar rat. In the rat the last generation of oogonia and multiplying prospermatogonia (M-prospermatogonia), frequently arranged in synchronized clusters, enters mitosis on about day 17 post conception (p.c.). The duration of the S-phase D-S-Duration and the minimal generation time Tmin of both kinds of "gonia" were determined by the method of labeled mitoses (22 female and 22 male fetuses derived from 11 pregnant rats were sacrificed from 2 to 22 h after a single i.p. injection of 3H-thymidine on day 17 p.c.). Three curves, derived from the labeled prophases, metaphases and the postmitotic descendents of oogonia and M-prospermatogonia--oocytes and primary transitional prospermatogonia (T1-prospermatogonia)--were evaluated. It was demonstrated that the curves as well as the calculated values of D-S-Duration and Tmin are very similar for oogonia and M-prospermatogonia. D-S-Duration ranged from about 10 to 12.5h (10 h read off from the curves of labeled metaphases), Tmin from 16.5 to 18 h (16.5 h read off from the curves of labeled metaphases).


Penetration of spermatozoon into the ovum and transformation of the sperm nucleus into the male pronucleus in the domestic fowl, Gallus gallus. The apex of the sperm head which has undergone the acrosome reaction comes in contact with the plasma membrane of the ovum. After the entire surface of the inner acrosomal membrane has come into close contact with the plasma membrane of the ovum, the two membranes fuse to form a continuous membrane. All parts of the spermatozoon that are devoid of plasma membrane penetrate into the ooplasm. As the head of the spermatozoon moves deeper into the ooplasm, the chromatin begins to disperse, and the head of spermatozoon is transformed into a large spherical nucleus with low electron density. At a later stage of the transformation, many small vesicles appear around the nucleus and subsequently fuse to form two continuous membranes. These membranes represent the male pronuclear envelope. The condensation of the chromatin occurs in places in the nucleus, so that the male pronucleus is formed. During the course of the formation of the male pronucleus, the subacrosomal rod and tail become detached from the head and disintegrate.


Lateral ocellar nerve projections in the dragonfly brain. The central projections of the lateral ocellar neurons of the dragonfly were examined using whole nerve cobalt iontophoresis, supplemented by sectioning of the nerve and brain for inspection in the light and electron microscopes. At E.M. level the presence of cobalt in filled axon profiles and cell bodies was confirmed by analysis of X-ray energy spectra in the microscope. The pathways, cell body sites and terminal arborizations of four large (7--25 micrometer diameter) lateral ocellar neurons are described. Two of these fibers arborize in the ipsilateral posterior neuropil of the protocerebrum and two cross the brain and arborize in the contralateral posterior neuropil. Within each half of the posterior neuropil, two spatially separated regions of ocellar input have been identified. These regions receive median ocellar input plus input from either the ipsi- or contralateral ocellus, but not both. The arborizations of the contralateral fibers are more extensive than those of the ipsilateral fibers. One of the contralateral neurons crosses the brain in the region of the protocerebral bridge giving off a collateral in that region before descending to the posterior neuropil. This collateral arborizes almost immediately in a region receiving input from arborizations of a number of small ocellar neurons (those less than 5 micrometer in diameter) from the ipsilateral ocellar nerve, together with small neurons from the median ocellar nerve, forming a region in each half of the brain which receives input from all three ocelli. The small lateral ocellar neurons associated with these arborizations have cell bodies adjacent to the lateral ocellar tracts.


Dopamine and noradrenaline neurons projecting to the septal area in the rat. The organisation of the catecholamine innervation of the rat spetal area was investigated by means of the glyoxylic acid fluorescence method in combination with dopamine uptake studies, lesions and retrograde tracing of horseradish peroxidase. The following catecholamine systems to the septum could be established: 1. The Locus Coreuleus Noradrenergic System. These axons are widespread in the septum forming a moderately dense innervation in the anterior hippocampus, the medial septal nucleus, the nucleus of the diagonal band, and the interstitial nucleus of the stria terminalis, and a sparse innervation in the lateral septal nucleus and the septofimbrial nucleus. 2. The Medulla Oblongata Noradrenergic System. This system originates in the A1, A2 or A3 cell groups, the axons forming a very dense innervation in the ventral part of the interstitial nucleus of the stria terminalis, a moderately dense innervation in the nucleus of the diagonal band and lateral septal nucleus, and a sparse innervation in the medial septal nucleus, the septofimbrial nucleus and the dorsal part of the interstitial nucleus of the stria terminalis. 3. The Mesencephalic Dopaminergic System. This system originates in the medial part of the A10 cell group, the axons forming two distinct terminal patterns. In the first type, smooth axons form pericellular arrangements around non-fluorscent neurons in the lateral septal nucleus. The second type is formed by fine-varicose axons which form a dense band around the fornix in the medial part of the lateral septal nucleus. 4. The Incerto-Hypothalamic Dopaminergic System. These axons most probably originate in cell bodies of the diencephalic A11, A13 and A14 cell groups, and are found in the lateral septal nucleus at the level of the anterior commissure.


Localization of monoamines in the forebrain of two salmonid species, with special reference to the hypothalamo-hypophysial system. In the salmon and trout aminergic cell bodies were found in the nucleus recessus lateralis (NRL) and the nucleus recessus posterioris (NRP), both of which are situated near the third ventricle. Three cell types could be distinguished. Type 1 produces a green and type 2 a yellow fluorescence. The former type probably contains dopamine and the later 5-hydroxytryptamine. Both types possess intraventricular protrusions in contact with the cerebrospinal fluid. The third cell type produces a less intense blue-green fluorescence; relatively few cells of this type have thick processes in contact with the ventricle. In addition, large fluorescent cells were found in the salmon, dorsal from the caudal part of the NRL. The various parts of the NRL and NRP are interconnected by thick bundles of nerve fibers; tracts leaving the nuclei could be traced for short distances only. The cells of the nucleus praeopticus (NPO), those of the medial part and to a much lesser extent also of the lateral part of the nucleus lateralis tuberis (NLT) have an aminergic innervation which probably originates form the NRL and/or NRP. All parts of the neurohypophysis contain many monoaminergic fibers, with aminergic material concentrated at the neuro-adenohypophysial interface. Fibers were not observed to penetrate the basal lamina. In the salmon and trout the fibers have a similar distribution, but differ in the intensity of fluorescence, being high in the salmon and low in the trout. Only in the trout have fluorescent cells been found in the adenohypophysis and very occasionally in the neurohypophysis. A number of these cells are basophilic and show a PAS-positive reaction.


Neuronal connectivity patterns in the compound eyes of Artemia salina and Daphnia magna (Crustacea: Branchiopoda). The neuronal types and patterns in the visual system of the species Artemia salina and Daphina magna have been studied with the Golgi method and electron microscopy. The lamina contains five classes of neurons: photoreceptor axons, monopolar, centrifugal, tangential and amacrine neurons. The terminals of the receptor axons are distributed in two (A. salina) or three (D. magna) layers. The dilated terminals have an extensive and wide array of fine branches. One axon from each ommatidium bypasses the lamina and terminates in the medula in A. salina. A. salina has four types of monopolar neurons, two of which are stratified, whereas in D. magna only two types are found, one of which is bistratified. Tangential T-neurons connect the lamina with the protocerebrum. D. magna has in addition one tangential T-neuron connecting both the lamina and the medulla with the protocerebrum. In both species monopolar-type centrifugal neurons connect the medulla and the lamina, whereas that of A. salina has a wide laminar distribution. Both species also have amacrine cells in the lamina. The medulla contains, besides those shared with the lamina, transmedullary neurons (two types in A. salina), amacrine cells and neurons originating in the protocerebrum. "Cartridge"-type synaptic compartments are lacking in the investigated species, although a periodic arrangement is discernible in the distal portion of the lamina of A. salina. The receptors from three types of specialized contacts in Artemia, one of which involves a dyad. D. magna has only one-to-one synapses. Neurosecretory fibres are absent in A. salina.


The effect of haemorrhage on the cell populations of the thymus and bone marrow in wild starlings (Sturnus vulgaris). Following the withdrawal of blood from the brachial vein of adult wild starlings (Sturnus vulgaris) changes in the cell populations within the bone marrow and thymus were observed over an eight day period. The packed cell volume, haemoglobin content and reticulocyte count of the peripheral blood was determined before and after haemorrhage. The maximum effect of the haemorrhage was observed in the bone marrow after four days when the population of small lymphocytes, and basophilic erythroid precursors were reduced to less than 1%. At the same time the percentage of another line of erythroid cells increased to 68%. This second erythroid lineage was the major erythroid line in the thymus, and again maximum representation occurred at 4 days post haemorrhage. After this the thymus became predominantly lymphoid and started to increase in size. The two erythroid lines are described and their status with regard to avian thrombocytes is also discussed. The peripheral blood had not attained the pre-haemorrhagic values for reticulocyte counts by eight days although the packed cell volumes and haemoglobin contents were similar.


An ultrastructural study of regeneration of minced smooth muscle in the vas deferens of the guinea-pig. Electron microscopic studies were made of the regeneration of minced smooth muscle of the vas deferens of the guinea-pig 3 days to 15 weeks after operation. At 3--5 days the mince contained degenerating smooth muscle cells and dedifferentiating cells showing characteristics of embryonic smooth muscle cells: numerous free ribosomes, well developed rough endoplasmic reticulum and Golgi apparatus with few peripherally placed myofilaments associated with dense bodies. During the first two weeks of regeneration, scattered cells surrounded by debris and collagen were separated by a large extra-cellular space. After three weeks, extracellular space was reduced to near normal values. Regenerating cells had a shorter length than normal cells, but during later stages of regeneration they showed an increase in diameter. Muscle effector bundles began to form after 2 to 3 weeks. Initially there were large gaps between the muscle cells, but at later stages of bundle formation, the extracellular space between the muscle cells was much reduced. From 3 weeks, arterioles appeared between the smooth muscle bundles in the regenerating areas. Regeneration of individual smooth muscle cells was complete by 15 weeks after the operation.


Reinnervation of regenerating smooth muscle cells in minced vas deferens of the guinea-pig. In adult guinea-pigs, a portion of the wall of the vas deferens was removed, minced and replaced. This caused muslce cells to dedifferentiate, divide and redifferentiate. Reinnervation of redifferentiating cells was followed using electron microscopy and histochemistry. Adrenergic nerves were first observed to re-enter the regenerating area 5 days after operation, and close contacts (within 20 nm) with muscle cells were first seen at 10 days. The total number of adrenergic nerves per 100 muscle cells reached contol values by 5 weeks, and by 15 weeks was higher than control levels. Cholinergic nerves first appeared in the regenerating area about 3--4 weeks after the operation. The total number of cholinergic nerves present had not reached control values even at 15 weeks, and no nerve muscle contacts within 20 nm were observed. The ratio of adrenergic to cholinergic nerves in the regenerating area was higher at 15 weeks than in control tissue.


The ultrastructure of the male and female prostate of Praomys (Mastomys) natalensis. I. Normal, castrated and ovariectomised animals. Male ventral and female prostates of Praomys (Mastomys) natalensis were examined with the electron microscope. The findings support and add to information obtained with the light microscope on tissues from normal, castrated and ovariectomised animals. Our results indicate that although the female prostate may be considered a homologue of the male ventral ventral prostate anatomically and histologically, there are differences in sub-cellular morphology and hormone dependence. Cells of the intact ventral prostate of the male are characterised by prominent dilated Golgi vesicles and electron-dense "mature secretory granules" seen in the apical region of the cell. In the cells of the female prostate these features are absent. These morphological differences reflect the influence of hormones upon the cells, as shown by the reduction of the dilated Golgi vesicles in the castrated male and conversely, their occasional presence in the cells of the oestrous female. Comparison of castrated and ovariectomised animals shows that the male ventral prostate is much more dependent on androgens than the female is on ovarian hormones. There are several modes of secretion in the male ventral and the female prostate. These are by acellular and cellular blebbing, by a variety of secretory vesicles into the acinar lumina, and by a system of "double walled" vesicles not previously described.


Podocytes in the blood vessel linings of Phoronis muelleri (Phoronida, Tentaculata). In several metasomal blood vessels of Phoronis muelleri myofilament-containing podocytes are the predominatnt cell-type. In some regions the podocytes can build a labyrinth resembling e.g. the glomerular epithelium of Enteropneusta and the axial organ of Asteroidea.


On the chromaffin cells in dog adrenal medulla; with special reference to the small granule chromaffin cells (SGC cells). Small granule chromaffin cells (SGC cells) were identified in the adrenal medulla of adult dogs. They were small in size and usually showed a high nucleo-cytoplasmic ratio. Cytoplasmic projections were occasionally observed in some of these cells. They contained a variable number of small secretory granules with diameters ranging from 70 to 300 nm, but mostly from 100 to 200 nm. The densities of the secretory granules were variable, ranging from highly dense to less dense. These adrenal SGC cells were rich in free ribosomes and polysomes, but were relatively poor in other cell organelles. Chromaffin cells which were intermediate in their characteristics (IM cells) between the SGC cells and the typical A and N cells were also identified. These IM cells contained both highly electron dense and less dense granules in various proportions. The IM cells were classified into two subgroups, according to the proportions of adrenaline type granules and noradrenaline type granules. One group resembled A cells (IM-A cells) and the other resembled N cells (IM-N cells). Light microscopic histochemical studies of A cells stained with the ammoniacal silver solution demonstrated that they contained a small number of darkly stained granules. Electron microscopic cytochemistry revealed that the electron dense granuls in the SGC cells, IM cells and A cells reacted positively with both the potassium dichromate solution at pH 4.1 and the ammoniacal silver solution.


The uterovaginal sperm host glands of the quail (Coturnix coturnix japonica): an ultrastructural and ultracytochemical study. Ultrastructural and ultrahistochemical studies were performed on the uterovaginal sperm host glands of the quail (Coturnix coturnix japonica). The proximal parts of the glandular necks are lined by a pseudostratified epithelium, consisting of high columnar ciliated cells and small, irregular shaped, basal cells. The true glandular epithelium is composed only of columnar cells with microvilli on their luminal end. A characteristic luminal feature is a large lipid droplet in the perinuclear region. In the subplasmalemmal region numberous tubular profiles are seen which could represent a cellular resorption system. To evaluate the absorptive capacity of the uterovaginal sperm host glands, tracer studies with HRP, ferritin, lanthanum and ruthenium red were undertaken. Since between 5 min and 3 h after injection no absorption could be found with the techniques mentioned, it is suggested that phagocytosis of spermatozoa by the glandular epithelium is not likely to occur.


Desmosomes: their occurrence between adjacent primary oocytes in polyovular follicles in the rabbit. In polyovular, primordial follicles in the rabbit, desmosomes are found between apposing oocyte surfaces. Morphologically these desmosomes correspond closely to those described in epithelia of vertebrates. The desmosomes alternate with other junctions which are probably gap junctions.


Antennal sensory system of Periplaneta americana L.: distribution and frequency of morphologic types of sensilla and their sex-specific changes during postembryonic development. The morphology of the antennal hair-sensilla of Periplaneta americana, their distribution and frequency on the antennal flagellum have been examined by transmission- and scanning-electron microscopy. The types of sensilla were distinguished with respect to physiologically relevant criteria such as wall structure and number of sensory cells. Among the sensilla of the antenna of the adult male, long, single-walled sensilla with four sensory cells (types sw B), Probably responsible for reception of sexual pheromones, are most frequent, representing about 54% of the antennal sensilla. About half of these sensilla are newly-formed at the imaginal ecdysis; the other half are derived from the shorter type sw B sensilla of the nymphal antenna. Short type sw B sensilla are present in all larval stages of both sexes and in adult females as well. During the imaginal ecdysis of males, however, the length of these sensilla increases to double that found in nymphs. Dendritic branches also increase in number. During postembryonic development, the number of sensory fibers in the antennal flagellum increases nearly 20-fold, from 14,000 in the first larval instar to about 270,000 in the adult male. The greatest increase, approximately 90%, occurs during the last developmental stage.


Extracellular microtubule-like structures in the retina of the rainbow trout: development, intercellular connectivity and reaction to vincristine. Extracellular microtubule-like structures (MLS) are described in the retina of the rainbow trout. They appear about 30 to 40 days after hatching, when the yolk-sac is consumed and the animal begins to swim and to nourish actively. They fill a widely branched system of extracellular clefts and spaces, and connect different cells and cell types, especially outer horizontal cells and bipolar cells. The MLS are not affected by the vinca alcaloid vincristine, although this drug penetrates into the MLS-filled space, as has been shown by the formation of intracellular, vincristine-induced tubulin paracrystals. The MLS are compared with other extracellular tubular structures described in other animal tissues. Their functional significance remains unclear.


Ultrastructural dynamics of the corpus allatum of Choleva angustata Fab. (Coleoptera, Catopidae). The ultrastructure of the corpora allata (CA) during postembryonic stages of Choleva angustata Fab. shows cyclic changes, in particular regarding the endoplasmic reticulum and the mitochondria. During the last larval instar, at the short transitory period which follows the cessation of alimentation, we observed a highly unusual appearance of the CA, characterized by intensive vacuolization. After this transitory period, the smooth endoplasmic reticulum (ser) disappears until the imago emerges. These observations along with our experimental data permit a physiological interpretation.


Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in long term cultures. Eel hemi-pituitaries were cultured in vitro on high or low sodium media, previously shown to affect differentially prolactin and growth hormone release. After 6 days culture, there were marked differences in the ultrastructure or both prolactin and growth hormone cells from the two groups. Morphometric data on the prolactin cells from SW-adapted eels showed a greater abundance of RER and paucity of secretory granules in cells from the low sodium medium. The size of the Golgi apparatus and the number of exocytosed secretory granules did not differ markedly between experimental groups, in contrast to previous findings on short-term cultures. Differences in the profile diameters of secretory granules are recorded between the experimental groups and the pattern differs markedly from that previously recorded for short-term cultures. The growth hormone cells from low sodium media were characterised by abundant, vesiculated RER, a prominent Golgi apparatus (in SW-adapted animals) and relatively few secretory granules. The activity of these growth hormone cells is in marked contrast to previous findings relating to short-term cultures. The shape and size of the non-granulated (stellate) cells of the RPD was again affected by the osmotic pressure of the medium.


The frontal ganglion of Periplaneta americana L. (Insecta): an electron microscopic and immunohistochemical study. The frontal ganglion, part of the stomatogastric nervous system, contains about 60 to 80 neurons, 25 to 30 micron in diameter. A well developed Golgi system, producing dense-core vesicles, lysosomes, multivesicular bodies and dense bodies are abundant. Glia elements are sparsely distributed. Many nerve fibres contain granules of different size and electron density. Five groups of fibres can be distinguished: Fibres with granules of about 200 nm (type A), fibres with granules of about 160 to 170 nm (type B), fibres with granules of about 80 to 100 nm (type C) and those with synaptic vesicles of 50 nm (type D) respectively. A fifth very small type contains neither vesicles nor granules. Special attention was paid to synaptic contacts. The divergent dyad seems to be the main type in the frontal ganglion. Frequently, neurosecretory endings are observed in presynaptic position. Immunocytochemical "staining" of neurosecretory material closely corresponds to the distribution of type A fibres, as observed electron microscopically. Immunoelectrophoresis of extracts from frontal ganglia with polyspecific anti-neurosecretion-serum reveals a single precipitation line, indicating that the immunocytochemical localization of neurosecretory material is due to reaction with a specific as well as a cross-reagent anitbody.