Kinetic response of human lymphoid cells to adriamycin-DNA complex in vitro. The lysosomotropic agent adriamycin-DNA complex was analyzed for its effect on cell cycle progression of human lymphoid cells in culture by means of pulse cytophotometry. Complexing to DNA slightly reduced the perturbation effects previously reported for adriamycin alone. The major kinetic response was a G2 block, the magnitude and duration of which was dependent on drug concentration and duration of treatment. When high drug concentrations were maintained for a prolonged period of time, an additional, completely reversible block in G1 phase or at the G1-S boundary was observed, accounting for two-step G2 accumulation curves. Cell age markedly influenced the magnitude of G2 accumulation in that treatment of cells in S and early G2 phase was most effective.


Morphological and clinical observations of patients with early bladder cancer treated with total cystectomy. In 21 cases, early bladder cancer was detected by urine cytology, although not by cystoscopy, and was treated by total cystectomy. The neoplasms, all transitional cell carcinomas of moderate to high degrees of anaplasia, were entirely in situ in 17 of the 21 patients; in 4, although mainly in situ, the tumors showed additional minimal microinvasion. Widespread mucosal involvement was demonstrated in every case by step-sectioning, and extension into the prostatic ducts occurred in 7 of the 19 male patients and into the mucosa of one or both distal ureters in 12 patients. Premalignant atypia of the mucosa was also widespread and direct intramucosal spread of cancer cells was a significant factor, particularly along the prostatic ducts and ureters. The duration of significant symptoms (follow-up for 9 years before cystectomy in several cases and for 8 years in 1 histologically proved case) suggests that the evolution of these tumors may be considerably longer than previously documented.


Membrane changes during urothelial hyperplasia and neoplasia. Normal mammalian urothelium has a complex pattern of morphogenesis and cell differentiation. The luminal face of the polyploid, superficial cells is limited by a morphologically unique plasma membrane with a highly ordered substructure. This membrane is a convenient marker for normal differentiation in the urothelium. In reversible, benign hyperplasia, following cytotoxic damage, the luminal face of the most superficial layer of cells is limited by an undifferentiated membrane which is comparable to the plasma membrane of normal basal cells. When the hyperplasia regresses, normal differentiation of the urothelium is reestablished, and the normal, specialized surface membrane is again produced. In both preneoplastic hyperplasia and early neoplasia induced by various carcinogenic regimens, a novel form of differentiation affecting the surface membrane is seen in some superficial cells. Microvilli form on their free luminal face and are limited by a membrane with an unusual, structured glycocalyx, which is not found in normal urothelium or benign hyperplasia. This glycocalyx also develops on some cells in transitional cell carcinoma of the bladder in man. This novel glycocalyx may be a morphological marker for neoplastic transformation in the urothelium.


Early lesions in experimental bladder cancer: experimental design and light microscopic findings. N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide (FANFT) fed to male and female Fischer rats at a dose of 0.2% of the diet induces lesions of the urinary bladder which progress from mild hyperplasia at 2 to 4 weeks, to moderate hyperplasia at 6 to 8 weeks, severe nodular and papillary hyperplasia at 10 to 14 weeks, and microinvasive carcinomas by 25 weeks as observed by light microscopy. Male Fischer rats fed FANFT for 2 to 4 weeks and then maintained on control diet show regression of the bladder lesions within 2 weeks to normal-appearing mucosa which persists through 50 weeks. Rats fed FANFT for 6 weeks show regression of the moderately hyperplastic epithelium to normal within 4 weeks after being placed on control diet which persists through 50 weeks. Rats fed FANFT for 8 to 10 weeks show regression of the hyperplastic bladder epithelium within 2 weeks of receiving control diet, but focal areas of mild hyperplasia are detectable through the 20th week of the experiment. By the 50th week the rats fed FANFT for 8 weeks all had moderate to marked hyperplasia, but the rats fed FANFT for 10 weeks had transitional cell tumors, one of which was invasive through the entire thickness of the bladder wall. The lesions present in rats fed FANFT for 12, 14, or 20 weeks continued to progress to invasive tumors (microinvasive or invasion of muscle) after the rats had been maintained on control diet through 50 weeks. Thus, the hyperplastic lesions developing through 6 weeks of FANFT administration appear to be reversible if FANFT is discontinued, but later lesions appear to be irreversible.


Early lesions in experimental bladder cancer: scanning electron microscopy of cell surface markers. The administration of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) at a dose of 0.2% of the diet to male Fischer rats results in the appearance of urinary bladder epithelial lesions progressing from hyperplasia to invasive transitional cell carcinomas. These progressive epithelial alterations have been observed by scanning electron microscopy at 2-week intervals with special attention paid to cells covering the luminal surface. After 2 to 4 weeks of FANFT administration there is mild pleomorphism and moderate swelling of the surface cells giving a cobblestone appearance. The normal pattern of microridges of superficial transitional cells is still present, although occasional cells by 4 weeks of FANFT feeding are covered by small uniform microvilli. By 6 weeks these changes are more extensive and more cells are covered with uniform microvilli. By 8 weeks, cells covered with pleomorphic microvilli are found in discrete foci, and by 10 weeks nodular or papillary lesions are present. The 2- and 4-week lesions regress to normal within 2 to 4 weeks of discontinuing FANFT administration, and the 6- and 8-week lesions regress toward normal in 4 to 6 weeks of being fed control diet. The 6-week lesions have regressed entirely to normal 44 weeks after FANFT was stopped, but the 8-week lesions have progressed to moderate or marked hyperplastic lesions by 42 weeks of control diet with pleomorphic cells covered with pleomorphic microvilli. The results of this study suggest that lesions are reversible up to and including 6 weeks of 0.2% FANFT in the diet, and that, by 8 weeks of 0.2% FANFT, irreversible lesions result which do not revert to normal after 42 weeks of control diet.


Changes in plasma membrane structure associated with malignant transformation in human urinary bladder epithelium. Integral membrane proteins are visualized as intramembrane particles (IMP; also called membrane-associated particles) at the cleaved surfaces of freeze-fractured plasma membranes. Topographical distributions of the IMP of urothelial cell membranes in normal human bladder and for a small series of low-grade noninvasive transitional cell carcinomas and invasive transtional cell carcinomas are shown to be significantly different. Using several statistical methods that test IMP topography via à vis the random (Poisson) hypothesis, it is demonstrated that IMP are mildly aggregated in plasma membranes of normal human urothelial cells and that, in noninvasive carcinomas, IMP aggregation is increased. In invasive transitional cell carcinomas, IMP are statistically nonaggregated and are in a random distribution in the plane of the membrane. IMP numerical densities are also altered in the course of neoplastic transformation. IMP are significantly increased in number in plasma membranes in human noninvasive transitional cell carcinomas but are similar to control values in invasive tumors. Loss of IMP and changes in IMP topography may be related to tumor invasiveness or they may represent an epiphenomenon.


Organ culture of normal and carcinogen-treated rat bladder. A system for organ culture of rat bladder in defined medium, without serum, is described. Transitional epithelial morphology of normal bladder was well maintained in Waymouth's Medium MB 752/1, while Ham's Medium F12 caused marked epithelial hyperplasia. Hyperplastic epithelial states induced by in vivo administration of the carcinogen, N-methyl-N-nitrosourea, were well maintained in vitro during organ culture in Medium MB 752/1. The combination of hydrocortisone and insulin markedly inhibited the proliferative response of normal bladder epithelium grown in Medium F12.


Sequential phenotypic changes in hyperplastic areas during hepatocarcinogenesis in the rat. Sequential phenotypic changes in hyperplastic areas of rat liver during N-2-fluorenylacetamide feeding were studied by enzyme and immunohistochemical methods combined with radioautography. Hyperplastic area showed a marked deficiency of beta-glucuronidase and serine dehydratase during their developing phase, the 6th through the 9th experimental weeks, and were fairly specifically labeled by injections of tritiated thymidine after partial hepatectomy performed at the 9th week. A sequential observation on these labeled hyperplastic areas revealed a considerable elevation of the levels of these marker enzymes in the majority of the labeled areas in 3 to 18 weeks after labeling. On the other hand, there was a small group of hyperplastic areas in which the enzyme deficiency persisted during the observation period. This type of lesion was generally larger than those showing enzymic maturation. Labeled cells were not detectable either in distinct hyperplastic nodules at late phase or in carcinomas. The metabolic regulation in the cells comprising hyperplastic areas was studied by checking the induction and repression of serine dehydratase after dietary stimuli. Serine dehydratase was not inducible in hyperplastic areas during the developing phase or in areas with persistent enzyme deficiency, but it was clearly induced and repressed in areas where there was an elevation of the endogenous enzyme level. The areas of hyperplasia with persistent enzyme deficiency and growth appeared to be more important than the ones of phenotypic maturation in relation to the later development of carcinoma. The phenotypic maturation in hyperplastic areas might represent reversion of altered cells towards normalcy from the condition related with neoplastic transformation.


Cytology and cytogenesis of neoplastic (hyperplastic) hepatic nodules. Cytochemical and electron microscopic investigations of neoplastic nodules induced in the rat liver by nitrosomorpholine or thioacetamide show that most neoplastic nodules are comprised of a rather heterogeneous cell population. At least four different types of altered hepatocytes can be distinguished: (a) "clear" glycogen storage cells with a dislocation and relative reduction of the granular endoplasmic reticulum; (b) "acidophilic" glycogen storage cells with a hypertrophy of the agranular endoplasmic reticulum; (c) fat-storing cells; and (d) basophilic cells poor in glycogen and rich in ribosomes. In addition, there are diverse intermediate cell types. The cytochemically demonstrable activity of glucose-6-phosphatase is reduced in most neoplastic nodules, but it may also be normal or even increased. The clear and the acidophilic cells precede the development of the neoplastic nodules by weeks and months. They usually form foci which are taken to be preneoplastic lesions. During the formation of neoplastic nodules and hepatocellular carcinomas originating for such foci the glycogen of the clear and the acidophilic cells is progressively reduced, whereas the number of ribosomes (basophilia) increases. This process, which may be accompanied by a transitory accumulation of fat, leads to the evolution of basophilic carcinoma cells. We conclude from these observations that the majority of the neoplastic nodules consist of a mixture of precancerous, definitely cancerous, and diverse intermediate cells. Neoplastic nodules in which basophilic cells prevail may already be carcinomas. Although the neoplastic nodules seem to be a frequent precursor of hepatocellular carcinomas, the latter may also develop without going through the nodule stage.


Sequential phenotypic and biochemical alterations during chemical hepatocarcinogenesis. Chronic exposure to chemical carcinogens induces in the target tissue a series of complex morphological and biochemical alterations that precede the appearance of overt cancer. Three types of experiments are described: (a) exposure of livers that had received subcarcinogenic doses of N-2-fluorenylacetamide to a subcarcinogenic dose of dimethylnitrosamine  resulted in a 100% yield of neoplastic nodules and hepatocellular carcinoma: (b) neither normal hepatocytes nor those obtained from neoplastic nodules were agglutinated by any of the lectins tested. This finding was also true for slowly growing cells from carcinomas, while those of rapidly growing carcinomas were agglutinated by several lectins; (c) analysis of nonhistone proteins isolated from neoplastic nodules demonstrated the appearance of many new species in euchromatin when compared with normal liver. Carcinomas demonstrated an even greater number of new species and they were demonstrated in heterochromatin as well.


Preneoplastic lesions of the human mammary gland transplanted into the nude athymic mouse. Morphologically normal lobules and atypical lobules postulated precancerous to ductal carcinoma were transplanted to test their biological behavior. Supravital staining disclosed the 1 to 4-mm microorgans. "Cleared" mammary fat pads of nude mice were optimal transplantation sites. Of the total of 217 transplants from 19 cancer-associated and 13 non-cancer-associated breast, 151 survived after 2 to 27 weeks. Of 61 surviving normal-appearing lobules from cancer-associated breasts transplanted without prior in vitro maintenance, 20 (30%) dedifferentiated, and of 48 surviving lobules from noncancerous breasts, 11 (20%) dedifferentiated. Fifteen of 28 histologically normal-appearing lobules (60%) obtained from cancer-associated breasts after age 50 dedifferentiated. Thirty of 36 atypical lobules isolated from the breast tissue were obtained from cancer-associated breasts and the 5 of those that dedifferentiated came from cancer-associated breasts. Twenty of 22 (90%) dedifferentiating transplants from cancer-associated breasts showed a vascularization response, whereas 3 of 7 (43%) from noncancer-associated breasts did so. If dedifferentiation in this experimental setting is indicative of a precancerous potential, the data on normal-appearing lobules obtained from cancer-associated breasts from women over age 50 suggest that these lobules carry the greatest precancerous potential. Such lobules probably belong to a type persisting after menopause and they may be hormonally autonomous. Such lobules might undergo further atypia in vivo and, eventually, cancerous transformation.


Angiogenic capacity of preneoplastic lesions of the murine mammary gland as a marker of neoplastic transformation. The angiogenic capacity of normal, preneoplastic, and neoplastic murine mammary tissues from high-tumor-incidence strains (C3H, C3H-AVV, C3H-AVVfB) was compared by implanting small biopsies on the iris surface in New Zealand White rabbits. Proliferation of iris blood vessels was studied by: (a) in vivo slit-lamp stereomicroscopy and fluorescein angiography; (b) colloidal carbon injection of the microvasculature; and (c) histological examination. Ninety % of mammary tumor implants elicited neovascular changes after 48 to 72 hr, regardless of their histological classification or the presence or absence of mammary tumor virus. Corticosteroid treatment did not abolish this response. In contrast, only 6% of implants from normal mammary glands caused any vasoproliferation. Thirty % of implants from premalignant hyperplastic alveolar nodules produced a pattern of vessel growth similar to tumor implants. D-2 line (hyperplastic alveolar nodule outgrowth) tissues, which have a high predicted tumor incidence, induced significantly more neovascular responses (p less than 0.002) than do morphologically and biochemically similar D-1 line tissues, which have a low predicted tumor incidence. These data suggest that angiogenic capacity is expressed during the malignant progression of murine mammary tissues; this property may provide a means for identifying those preneoplastic tissues that are most at risk for cancer.


Prophylaxis of early preneoplastic lesions of the mammary gland. Daily treatment (for 12 to 14 months) of 2-month-old nulliparous or 8-month-old multiparous C3H/HeJ mice with 0.1 mg of 2-bromo-alpha-ergocryptine (CB-154) or 6-methyl-8-beta-ergoline-acetonitrile, efficacious inhibitors of prolactin secretion, markedly reduced the incidence of spontaneous mammary hyperplastic nodules and mammary tumors. CB-154 appeared to be more effective than 6-methyl-8-beta-ergoline-acetonitrile in suppressing the incidence of mammary tumors; the ergot virtually prevented the appearance of mammary tumors in nulliparous mice. Daily treatment of 5-month-old estrogen-treated, ovariectomized-hysterectomized C3H/HeJ mice for 12 months with CB-154 also significantly reduced the incidence of hyperplastic nodules and mammary tumors when compared with ovariectomized-hysterectomized mice treated with steroid alone. Daily treatment of multiparous C3H/HeJ mammary tumor-bearing mice with CB-154 or 6-methyl-8-beta-ergoline-acetonitrile generally failed, however, to promote regression of the mammary tumors. Thus significant prophylaxis of early preneoplastic lesions by drug-induced hormone (prolactin) suppression, resulting in a marked reduction in mammary tumor incidence, has been demonstrated in this study.


Precursor lesions of bronchogenic carcinoma. Clinical investigations and experimental studies concerned with preneoplastic and early neoplastic lesions in the respiratory tract are discussed. The occurrence of preinvasive states of carcinoma in the bronchus has been recognized for over 20 years. Histopathological and cytological studies suggest that it might become possible to identify even earlier preneoplastic precursor lesions provided the proper tissue or cell markers can be found. Experimental models that could be useful in establishing a better understanding of the evolution of the neoplastic diseases in the respiratory tract are now available.


Early proliferative changes in intestinal cells. Early lesions in the colonic mucosa of humans and rodents are characterized by similar proliferative changes within their epithelial cell population. Progressive phases of abnormal cell development appear during the evolution of neoplastic transformation in colonic cells of rodents exposed to chemical carcinogens and in humans highly susceptible to gastrointestinal cancer. Identification and classification by phenotype of cells of these individuals at increased risk for colon cancer are leading to new methods to improve the detection and diagnosis of neoplasia in high risk individuals and families. An analytical system of precise numerical definitions is aiding an approach to modify the evolution of advanced stages of neoplasia.


The precursor tissue of ordinary large bowel cancer. Hyperplastic polyps are 10 times as common as adenomas and must be distinguished from them since they are unrelated as a precursor tissue to either adenomas or carcinomas. Only adenomas are relevant to the development of the common moderately and well-differentiated large bowel cancer. Depending on three related factors (increasing size, a sessile rather than pedunculated mode of growth, and a villous rather than tubular microscopic architecture), one may find minute (1 to 2-mm) or microcancer with increasing frequency in adenomas. However, despite unlimited opportunity to do so, minute or microcancer has not been observed in normal mucosa, i.e., unassociated with adenomatous tissue. The same findings obtain in familial polyposis. In this condition, in grossly normal areas of mucosa, adenomas (but not carcinomas) as small as one or two crypts have been found. Direct one-step transformation from normal crypt cells to cancer, without formation of adenomatous epithelium, does not seem to be the usual pathway.


Characteristic complements of nuclear nonhistone proteins in colonic epithelial tumors. Nuclei were isolated from colonic epithelial tumors induced in rats by the administration of 1,2-dimethylhydrazine. The nuclei were fractionated according to buoyant density by centrifugation in discontinuous sucrose density gradients. Nuclei differing in density differ in size, nonhistone protein-to-DNA ratio, and DNA synthetic activity. Their distribution in a density gradient also reflects their histological localization in the layers of the intestinal mucosa, as judged by the nuclear capacity for [3H]thymidine incorporation into DNA in vivo. Different nuclear classes isolated from the tumors contain characteristic complements of nuclear nonhistone proteins. Particularly striking accumulations of two protein classes with molecular weights of ca. 44,000 and 62,000 occur during carcinogenesis. These proteins are not uniformly distributed throughout all nuclear classes derived from the tumors. They are not at all prominent in normal colonic epithelial nuclei or in epithelial cells surroundign the tumors, or in the liver nuclei of animals treated with 1,2-dimethylhydrazine. Procedures for the differential extraction of these protein classes are described. Similar nuclear proteins have been detected in human colonic tumors and in a human cell line (HT-29) derived from an adenocarcinoma of the colon. The selective accumulation of such proteins in colonic tumor nuclei may have diagnostic value.


Biochemical changes in preneoplastic rodent intestines. Two enzymes were examined as potential indicators of early precancerous changes. Ornithine decarboxylase, an enzyme normally associated with rapid cell division, is low in the rapidly dividing, cancer-susceptible colon. The level of this enzyme was also very high in the nondividing cells of the small intestines. Administration of an intestinal carcinogen, dimethylhydrazine, led to a large increase in colonic ornithine decarboxylase but did not affect the enzyme in liver. A liver carcinogen, acetylaminofluorene, induced manyfold increases in ornithine decarboxylase of the liver but not of the colon. Studies of thymidine kinase of the gut showed that this enzyme changed quantitatively and qualitatively throughout the life of the animal, from fetal rat to newborn and adult. The tumor enzyme has many fetal-like properties. Long-term treatment with dimethylhydrazine led to changes in thymidine kinase reminiscent of the fetal enzyme. Short-term treatment caused sharp increases in the thymidine kinase of nondividing cells of the jejunum and the proximal end of the colon; similar changes in the distal end of the colon were slower in appearing and less pronounced.


Morphological lesions associated with human primary invasive nonendocrine pancreas cancer. In 227 cases of human pancreas cancer (100 pancreatectomy specimens and 127 autopsies), pancreas duct epithelium not involved by invasive cancer was examined. Pancreas duct epithelium from 100 autopsies of patients with nonpancreatic cancer, matched by age and sex to the pancreas cancer autopsy cases, was used for control studies. The prevalence of squamous metaplasia, pyloric gland metaplasia, mucous hypertrophy, and focal epithelial hyperplasia was not greatly different in the two groups. Ductal papillary hyperplasia was three times more prevalent in pancrease cancer than in controls. Marked atypia occurred in 20%, and carcinoma in situ, in 18% of the pancreas cancer cases, but neither change was seen in the control cases. It is possible that focal epithelial hyperplasia was a precursor change but that it was overgrown by the cancer. Papillary hyperplasia could not be properly evaluated as a precursor lesion because of duct obstruction, but practically all cases of marked atypia and carcinoma in situ occurred in papillary lesions. Marked atypia and carcinoma in situ, by analogy to other cancers, would appear to be precursor lesions, and their presence in association with invasive cancer lends hope to the possibility that there is a significant, recognizable, in situ phase of the disease before invasive cancer occurs.


Approaches to prevention of epithelial cancer during the preneoplastic period. The development of epithelial cancer is a disease process that takes many years to reach its final, invasive stage in humans. This disease process has the potential to be controlled by physiological or pharmacological means during its preneoplastic stages. Mechanisms whereby the progression of preneoplastic lesions can be stabilized, arrested, or reversed are discussed. Pharmacological enhancement of such mechanisms by synthetic vitamin A analogs (retinoids) offers a possible means for prevention of invasive epithelial cancer.


Differential inhibition of embryonic cell aggregation by cultured human cells with "malignant" of "normal" characteristics. The effect that cultured human cells have on chick embryonic neural retina cell aggregation was examined. Different types of human cultured cells inhibited aggregation of chick neural retinal cells to differetn degrees when mixed at a human cell:retina cell ration of 1:60. It appeared from the eleven cell lines studied that cells with "malignant" characteristics inhibited retinal cell aggregation to a greater extent than those with more "normal" characteristics. The assay could be used as a further test for abnormality of cell types and also as a method for studying the interactions of malignant cells with cultured cells.


Effect of vincristine on sister chromatid exchanges of normal human lymphocytes. In vitro analysis of the mitosis from 3-day lymphocyte cultures from two normal males and two normal females treated with bromodeoxyuridine and stained with acridine orange after addition of vincristine during different times demonstrated that vincristine inhibits the progression of the second division. When the second division progressed, the number of sister chromatid exchanges was significantly decreased. Vincristine could act by inhibiting the copying of DNA. A dosage effect is demonstrated when vincristine is added to the cultures during the first 24 hr.


The effect of Corynebacterium parvum in combination with 5-fluorouracil, L-phenylalanine mustard, or methotrexate on the inhibition of tumor growth. Previous reports from this laboratory have demonstrated conclusively that cyclophosphamide administered asynchronously with Corynebacterium parvum (CP) results in greater C3H mammary tumor inhibition than that observed with either agent alone. An analysis of this combination has revelaed that the chemotherapeutic component contributes more significantly to tumor inhibition than does the immunotherapeutic one. This study was conducted to investigate the inhibition of C3H mammary tumors by other chemotherapeutic agents when used with CP. The results have demonstrated that 60 mg of cyclophosphamide per kg, 90 mg of 5-fluorouracil per kg, and 10 mg of L-phenylalanine mustard per kg administrated weekly have similar tumor-inhibiting properties. The addition of CP enhanced the tumor-inhibiting properties of each agent but to differing degrees. The effect of the immunopotentiator when used in combination with alkylating agents was greater than that seen when it was used with the antimetabolite 5-fluorouracil. The tumor inhibition observed when cyclophosphamide was administered asynchronously with CP was significantly greater than that observed when L-phenylalanine mustard was similarly used. Of particular interest was the finding that the addition of CP to a combination of chemotherapeutic agents resulted in no greater tumor growth inhibition than that which occurred when CP was used along with the most effective single agent in the combination. The data have indicated that, contrary to clinical impression, there is no evidence that CP through its toxicity-sparing effect permits the utilization of larger doses of chemotherapy. Consideration has been given to the mechanisms that might account for the differences in tumor growth inhibition encountered when CP was used with different chemotherapeutic agents.


Antitumor activities of newly synthesized N4-acyl-1-beta-D-arabinofuranosylcytosine. New derivatives of 1-beta-D-arabinofuranosylcytosine were synthesized and their antitumor activities were tested against mouse leukemia L1210. Among the 50 compounds investigated, a series of N4-acyl derivatives with long-chain saturated fatty acids were found to be highly active. The most active derivatives were N4-stearoly-1-beta-D-arabinofuranosylcytosine, which was administered in the form of suspension, and N4-behenoyl-1-beta-D-arabinofuranosylcytosine given in the form of solution. They were superior to the parent compound, 1-beta-D-arabinofuranosylcytosine, in that smaller dosages exhibited strong activities regardless of the treatment schedule, and they were also resistant to cytidine deaminase.


Mammary neoplasia in a closed beagle colony. The incidence rate of mammary neoplasia in a large colony of beagles and the relationship to internal skeletal and/or liver radiation, age, relatively late ovariectomy (4 years and older), endometritis, parity status, and adrenal weight were examined. Of these various factors, age was the only condition that was clearly correlated with changes in the mammary tumor incidence. The rate became significant at approximately eight years of age and increased progressively throughout the older age classes. Among the female dogs, the incidence of mammary cancer was higher than that of any other form of spontaneous malignancy.


Flow microfluorimetric analysis of sensitive and resistant leukemia L1210 following 1-beta-D-arabinofuranosylcytosine in vivo. Two groups of BALB/c X DBA/S F1 mice with sensitive and 1-beta-D-arabinofuranosylcytosine-resistant L1210 ascites, respectively, were used to study the cytokinetic effects of a single dose of 750 mg of 1-beta-D-arabinofuranosylcytosine per kg. Sequential DNA histograms, labeling indices, and mitotic indices were obtained from each of five mice at timed intervals from 0 to 72 hr. Each histogram was obtained by flow microfluorimetry, using the DNA-specific fluorochrome, mithramycin. The histograms were then integrated for cell cycle analysis. Cytokinetic perturbations occurred in both groups, but they were greater in the sensitive population where there was a relative accumulation of cells, mainly in early S phase, at 16 hr. This was followed by a relative depletion of S-phase cells. In the resistant population, there were relative accumulation of cells in early S phase at 8 and 24 hr and in mid-S phase at 32 hr, but there was no subsequent relative depletion of S-phase cells. The labeling index was rapidly reduced in both groups but was recovered in the resistant population within 4 hr. In the sensitive population, there was a transient rise in the labeling index at 8 and 16 hr. Sensitive and resistant populations of L1210 cells were rapidly and reliably distinguished by DNA content cell cycle analysis of a single sample taken between 24 and 72 hr following a large dose of 1-beta-D-arabinofuranosylcytosine. This technique has potential clinical application in the rational design and monitoring of chemotherapy.


Determination of carbocyanine dye-binding polyanions in malignant and nonmalignant disease states. Cancer-related changes in the serum seromucoid fraction are well known. Last year Woodman published an interesting carbocyanine dye binding method for determination of serum carbohydrate polyanions in sera of normal, traumatized, and tumor-bearing mice. The usefulness of this method for clinical practice has been investigated in this study. Carbocyanine dye-binding polyanion (CPA) and the sialidase-sensitive fraction of this polyanion (SPA) have been determined in sera of 705 human subjects including healthy normal individuals and patients suffering from a broad spectrum of malignant and nonmalignant disease states. Overall, in malignant diseases the CPA and SPA values, in mg pectin equivalents per liter (mean +/-2 S.D.) (292 +/- 111 and 135 +/- 68, respectively) were significantly higher than in the serum from normal controls (166 +/- 33; 74 +/- 18) and patients hospitalized with a variety of nonmalignant disease (195 +/- 56; 92 +/- 36). The highest CPA and SPA values were found in gynecological (331 +/- 117; 149 +/- 69), bronchial (294 +/- 72; 137 +/- 51), and gastrointestinal cancers (316 +/- 111; 154 +/- 69). Elevated CPA values were found in 59.9% and elevated SPA values in 52.8% of patients suffering from malignant diseases. Successfully, radically treated cancer patients with no detectable residues or metastases for at least 1 year had values (186 +/- 39; 76 +/-24) almost within the normal ranges (93 to 250 mg pectin equivalents per liter for CPA and 35 to 120 mg pectin equivalents per liter for SPA).


Lipid hydroperoxide activation of N-hydroxy-N-acetylaminofluorene via a free radical route. The data presented here demonstrate that linoleic acid hydroperoxide in the presence of methemoglobin or hematin activated the carcinogen N-hydroxy-N-acetyl-2-amino-fluorene via the nitroxyl free radical intermediate into 2-nitrosofluorene and N-acetoxy-N-acetyl-2-aminofluorene. Ascorbate inhibited the activation, in which case the free radical intermediate was replaced by the ascorbate free radical. On the basis of optical kinetics, we have established that the rate of linoleic acid hydroperoxide decrease paraleled the rate of N-hydroxy-N-acetyl-2-aminofluorene decrease and also the rate of 2-nitrosofluorene increase. The stoichiometry of the reaction was such that, for every 2 linoleic acid hydroperoxide molecules consumed, 2 N-hydroxy-N-acetyl-2-aminofluorene molecules were oxidized and 1 2-nitrosofluorene and 1 N-acetoxy-N-acetyl-2 aminofluorene molecule was formed.


Folate deficiency in the livers of diethylnitrosaminetreated rats. The effects of diethylnitrosamine on the metabolism of folic acid and related compounds in rat liver were investigated. The administration, in the drinking water, of diethynitrosamine to rats for 3 weeks led to decreased hepatic levels of folate, S-adenosylmethionine, and 5-methyltetrahydrofolate:homocysteine methyltransferase. Liver methylenetetrahydrofolate reductase levels were unaffected by administration of diethylnitrosamine. The polyglutamate fraction of hepatic folates obtained from rats treated with diethylnitrosamine for 3 weeks prior to injection with [3H]folate contained less radioactivity than did the polyglutamate fraction obtained from the livers of control rats treated with [3H]folate alone. Similarly, the polyglutamate folate fraction of rat livers that were simultaneously perfused with both diethylnitrosamine and [3H]folate contained less label than the polyglutamate fraction of rat livers perfused with [3H]folate only. Livers perfused with [2-14C]histidine and diethylnitrosamine produced more formiminoglutamate and less CO2 than livers treated with [2-14C]histidine only. The changes noted in the hepatic folate metabolism of diethylnitrosamine-treated rats resemble those seen in the livers of methyl-deficient rats.


The effect of phenobarbital on cyclophosphamide antitumor activity. We have used the spleen colony assay system and survival duration studies in male DBA/2 mice with P388 leukemia to study the effects of microsomal enzyme induction by phenobarbital on the antileukemic activity and bone marrow toxicity of cyclophosphamide. Phenobarbital drinking water (0.5 mg/ml) was given for 7 days prior to cyclophosphamide (10 to 200 mg/kg i.p.). Average daily phenobarbital intake per mouse was 1.25 mg (equivalent to 4 mg/kg/day human dosage). Dose-response curves with and without phenobarbital pretreatment showed a constant 90% (1-log) reduction in the toxicity of cyclophosphamide to leukemic colony-forming units, whereas enzyme induction had no effect on the toxicity of the drug to normal bone marrow colony-forming units. Parallel survival studies confirmed the 1-log diminution in the antileukemic activity of cyclophosphamide in phenobarbital-pretreated mice. This phenobarbital-induced change in the antitumor activity of cyclophosphamide appears explainable on a pharmacokinetic basis. The Friedman and Boger assay for plasma alkylating metabolites showed that the reduction in the area under the plasma metabolite curve caused by enzyme induction exactly predicted the observed reduction in cyclophosphamide antitumor effect.


The effect of allopurinol on cyclophosphamide antitumor activity. We have used the spleen colony assay system and survival duration studies in male DBA/2 mice with P388 leukemia to study the effects of allopurinol pretreatment on the antileukemic activity of cyclophosphamide and its bone marrow toxicity. Allopurinol drinking water (0.5 mg/ml) was given for 7 days prior to cyclophosphamide (10 to 200 mg/kg i.p.). Average daily allopurinol intake per mouse was 1.25 mg (equivalent to 4 mg/kg/day human dosage). Dose-response curves with and without allopurinol pretreatment showed an almost constant 0.9-log increase in the toxicity of cyclophosphamide to leukemic colony-forming units, whereas allopurinol had no effect on the toxicity of cyclophosphamide to normal bone marrow colony-forming units. Parallel survival studies revealed no difference in the antileukemic activity of cyclophosphamide as a result of allopurinol pretreatment. The allopurinol-induced change in the antitumor activity of cyclophosphamide as seen in the spleen colony assay was not explainable on the pharmacokinetic basis. Flow microfluorometric analysis of P388 leukemia tumor cell cycle parameters revealed no change in the blockading effects of cyclophosphamide as a result of allopurinol preexposure. Although we have failed to explain the underlying mechanism of this drug interaction, our data suggest that allopurinol may increase the antitumor activity of cyclophosphamide without increasing its bone marrow toxicity.


Histological comparison of the growth of rat bladder carcinoma R-4909 observed for two years in vitro and in vivo. A transplantable bladder tumor (Chapman R-4909) of the rat, when first received in our laboratory, grew with a complex histopathology. The predominant component was transitional cell carcinoma, but there were foci of keratinization, including pearl formation, and foci of a less well-defined cystic appearance. We report here observations made during the first 2 years of an ongoing study on the divergent histopathology of R-4909 under several conditions of propagation. During the entire period, the tumor has been maintained by serial passage in rats (Fischer 344) and by serial passage in vitro. At intervals, cells of the tissue culture series were inoculated into rats to compare the histopathology of animal- and culture-passed strains. We obtained several clones from the stock cultures and these also were maintained continuously in vitro. At intervals, cells from two of these lineages, clone A and clone B, were inoculated into rats. After 2 years, cells maintained in stock culture, on injection into new rats, produced growths similar to the original in that all three epithelial patterns, transitional, squamous, and adenomatous, were perpetuated. In contrast, the tumor passed exclusively in vivo lost its squamous component completely. It became anaplastic, with tissue architecture almost entirely adenomatous and cystic. Unlike the stock tissue culture line, the clonal isolates following prolonged culture produced adenomatous tumors only. In a related preliminary study, we inoculated into rats R-4909 cells that had been cultivated for up to 2 months under aerobic and anaerobic conditions. Tumors grew in most of the animals, and those of the aerobic group were more cystic than the others.


Prolonged tumor dormancy by prevention of neovascularization in the vitreous. Tumors release a diffusible substance that stimulates neovascularization. To study the neovascularization that occurs in diabetic retinopathy, we implanted V2 carcinomas and mouse ependymoblastomas into the vitreous of experimental animals. In the vitreous, unlike previous sites, the tumors failed to stimulate neovascularization. They grew for weeks as small, unvascularized, three-dimensional aggregates of cells. Explosive growth into a large, vascularized mass occurred when the avascular tumors reached the retinal surface. The vitreous proved to be a valuable model for observing the in vivo growth of small, solid tumors. Xenografts survived for months without evidence of immune rejection. The consequence of the prolonged avascular state is the restriction of tumor size. The normal vitreous may act to inhibit capillary proliferation. An understanding of the mechanism for maintaining the avascular state may lead to therapeutic blockade of neovascularization. This would be important in the management of diabetic retinopathy and neoplasia.


The sensitivities of human and murine hemopoietic cells exposed to cytotoxic drugs in an in vivo culture system. An agar diffusion chamber technique has been used to measure the sensitivities of human and murine hemopoietic colony-forming cells to cytotoxic drugs. The cells were held in i.p. diffusion chambers and exposed to the cytotoxic drugs by i.v. injection of the host mice. This method allows some account to be taken of the continuous changes in activity during the metabolic degradation of the drug. To determine how far this system provides a valid measure of the sensitivity of the cells in hemopoietic tissue, the responses of mouse bone marrow exposed to the drugs in situ in the donor mouse were compared with those of mouse cells exposed in diffusion chambers. The dose-response curves for cyclophosphamide and 5-fluorouracil were exponential in all cases. Exponential survival curves were also seen when human and mouse colony-forming cells were exposed to vinblastine or methotrexate in diffusion chambers. The plateaus seen when mouse cells were exposed to these drugs in situ could, however, be regained by omitting agar from the chambers during the exposure period. The results indicate that there are differences between the sensitivities of human and mouse marrow cells to cytotoxic drugs and that any extrapolation from mouse to humans must be viewed with caution.


Spontaneous extracellular synthesis of DNA released by human blood lymphocytes. Human lymphocytes were shown to release, in vitro and in the absence of any stimulation, a complex containing DNA. It has also been reported that the release process is unrelated to cell death and is regulated by a homeostatic mechanism. Some properties of the extracellular DNA were investigated. When a phosphorylated precursor was added to the cell-free supernatant, the DNA recovered from the medium was labeled. Evidence that DNA lebeling represented true precursor incorporation and not simple attachment was obtained from nearest neighbor analysis data. When [alpha-32P]thymidine triphosphate was added to the supernatant and the labeled DNA was completely hydrolyzed to 3'-deoxyribonucleotides, radioactivity was found in all four nucleotides. Although the exact kind of synthesis cannot be determined at this stage, the possibility of a terminal transferase system in which the enzyme would merely add a nucleotide at the end of the chain was eliminated since comparative digestion with DNase and venom phosphodiesterase showed that labeling was located along the whole length of the chain. Precursor incorporation into the DNA was inhibited by DNase, RNase, Pronase, and actinomycin D. This extracellular synthesis was not affected by cell death rate. The renaturation curve of the extracellular [3H]DNA synthesized in the cell-free medium showed a lack of gene reiteration suggesting a preferential synthesis of unique sequences.


A galactosidase immunosorbent test for carcinoembryonic antigen. A galactosidase immunosorbent test for carcinoembryonic antigen (CEA) is described in which the amount of galactosidase adsorbed to a cellulose disc is a hyperbolic function of CEA concentration. Thus, molecules with CEA-like activity can be characterized by mathematical analysis of data obtained from the galactosidase immunosorbent test. By such analysis, CEA-reactive molecules in normal human plasma were distinguished from normal cross-reacting antigen and from authentic CEA. Variation of the amount of antibody-enzyme conjugate used in the galactosidase immunosorbent test permitted CEA-reactive material in plasma of a patient with rectal carcinoma to be antigenically distinguished from the CEA-reactive material in urine of a patient with bladder carcinoma. The galactosidase immunosorbent test is a useful tool for analysis of CEA-reactive molecules.


Application of a galactosidase immunosorbent test to carcinoembryonic antigen in plasma. The galactosidase immunosorbent test for carcinoembryonic antigen is simple to perform, uses stable reagents, does not require radioactive reagents, and is adaptable to large numbers of samples. Concentration of carcinoembryonic antigen in sera or plasma was determined by the galactosidase immunosorbent test and by the Egan-Todd double antibody assay (93% agreement), indirect Z-gel (83% agreement), and direct Z-gel assay (ps = 0.97). The galactosidase immunosorbent test has potential as a clinically useful nonisotopic assay for carcinoembryonic antigen.


In vitro correlates of transformation in C3H/10T1/2 clone 8 mouse cells. Various potential in vitro correlates of malignancy were studied in four chemically transformed C3H/10T1/2 Clone 8 mouse cell lines and were compared with controls cells. The degree of tumorigenicity was best predicted by the relative plating efficiencies of the morphologically transformed cells in soft agar. All transformed cells also showed an increase in extracellular fibrinolytic activity which may be an additional marker for transformation. Intracellular fibrinolytic activity and loss of 125I-labeled cell surface protein (M.W. 250,000) were not correlated with morphological transformation or tumorigenicity in these cells.


Colon carcinogenesis in germ-free rats with intrarectal 1,2-dimethylhydrazine and subcutaneous azoxymethane. The effect of intestinal microflora on colon carcinogenesis by 1,2-dimethylhydrazine and azoxymethane was studied, with the use of germ-free and conventional female Fischer rats. At 7 weeks of age, germ-free and conventional rats were treated with 20 weekly intrarectal 1,2-dimethylhydrazine (20 mg per kg body weight per week) or subcutaneous azoxymethane (10 mg per kg body weight per week) doses and were autopsied 15 weeks later. Tumors were induced in the small intestine and colon of germ-free and conventional rats treated with intrarectal 1,2-dimethylhydrazine; the number of rats with colon tumors and the multiplicity of tumors were decreased in germ-free rats, compared with conventional animals. Azoxymethane given subcutaneously increased the incidence and multiplicity of colon tumors in germ-free rats, compared with conventional controls. It is concluded that the intestinal microflora alter the carcinogenic and/or cocarcinogenic effect of different compounds in the large intestine.


N-nitroso-bis(2-acetoxypropyl)amine as a further pancreatic carcinogen in Syrian golden hamsters. N-Nitroso-bis(2-acetoxypropyl)amine, a possible beta metabolite of N-nitroso-di-n-propylamine, was shown to be a potent carcinogen in the Syrian golden hamster. After a single s.c. treatment, the pancreas was the most affected organ, followed by the liver, respiratory tract, and kidneys. However, repeated application resulted in a higher incidence of neoplasms of the respiratory tract than of the pancreas and kidneys. The effect of N-nitroso-bis(2-acetoxypropyl)amine on toxicity, target tissues, and carcinogenicity was similar to that of N-nitroso-bis(2-hydroxypropyl)amine. The assumption that these two compounds may have similar metabolic pathways was confirmed; N-nitroso-bis(2-acetoxypropyl)amine was readily deesterified to N-nitroso-bis(2-hydroxypropyl)amine in vivo and in vitro.


Effect of adriamycin on DNA, RNA, and protein synthesis in cell-free systems and intact cells. The effect of adriamycin on DNA, RNA, and protein synthesis was investigated in cell-free systems and intact cells. In studies with purified mammalian cell enzymes, adriamycin produced a greater inhibition of DNA-dependent DNA polymerase than of RNA polymerase. The extent of inhibition of both these enzymes was decreased by increasing the concentration of the DNA template in the reaction mixture. In studies with isolated nuclei, adriamycin was also a more potent inhibitor of DNA synthesis than RNA synthesis. However, with intact cells, adriamycin inhibited both DNA and RNA synthesis to about the same extent. The inhibition produced by adriamycin on RNA synthesis in intact cells was greater than that observed in the cell-free systems. Adriamycin inhibited protein synthesis in a cell-free system consisting of polyribosomes, transfer RNA, and enzymes but did not inhibit protein synthesis in intact cells. These differences in the pattern of inhibition may be due to biotransformation of the drug and/or preferential binding to chromosomal DNA in the intact cell.


Separation and characterization of transcriptionally active and inactive nuclear subfractions of AKR mouse embryo cells. This study was initiated with the objective of separating and characterizing two or more nuclear subfractions, which could then be compared with respect to their relative propensity for binding carcinogenic polycyclic aromatic hydrocarbons. Nuclei were isolated from cloned AKR-2B mouse embryo cells, which are susceptible to transformation by chemical carcinogens. The nuclei were mechanically sheared and subfractions were separated by sedimentation through a 0.17 to 1.7 M sucrose gradient. When the cells were treated with [3H]uridine for 30 min, most of the label incorporated into RNA was recovered in the top region of the gradients, which represented Nuclear Subfraction I. The majority of the chromatin DNA, however was localized in the bottom region (Subfraction II) and the pellet (Subfraction III). Precipitation (with CaCl2) of the rapidly labeled RNA of Subfraction I along with the chromatin DNA suggested that the label was present in nascent RNA chains still attached to the chromatin. Thus, the transcripitionally active chromatin seemed to be localized in Nuclear Subfraction I. The chromatin of Subfraction I was also the best template for RNA synthesis in vitro with exogenous bacterial polymerase. The protein and RNA content of subfraction I was greater than that of the other two subfractions and whole chromatin. Electron microscopy revealed the presence of membrane material in Subfraction I and II, with little such material in Subfraction III. Subfraction I differed from Subfractions II and II and whole chromation with respect to thermal denaturation of the DNA and histone composition (as determined by gel electrophoresis). The acidic protein composition (as determined by gel electrophoresis) differed for the chromatin of all three nuclear subfractions.


Binding of polycyclic aromatic hydrocarbons to transcriptionally active nuclear subfractions of AKR mouse embryo cells. The objective of this study was to examine the binding of carcinogenic polycyclic aromatic hydrocarbons in well-characterized nuclear subfractions from transformable cells in culture. A cloned line of AKR mouse embryo cells was exposed to culture medium containing [3H]-3-methyl-cholanthrene (MC) (0.4 mug/ml) 670 Ci/mole). Cellular uptake and nuclear binding were determined after 4 hr of exposure. The addition of unlabeled MC up to 10 mug/ml did not cause reduction of [3H]MC cellular uptake or nuclear binding. From 2 to 5% of the total cellular MC was localized in the nuclei. All nuclear subfractions obtained from mechanically sheared nuclei and separated on sucrose gradients showed some MC binding; however, a high-affinity, high-specific-activity binding of MC was associated only with the slower-sedimenting component shown to represent that fraction of nuclear chromatin that is transcriptionally active. Conditions that caused the precipitation of this chromatin also resulted in the precipitation of the radioactive compound, thus suggesting that the MC was physically bound to the chromatin. Unlabeled MC (10 mug/ml) saturated this high-affinity MC binding to the transcripitionally active chromatin but did not saturate the binding to the other nuclear fractions. The binding of another potent carcinogen, [3H]-1,2,5,6-dibenzanthracene, and the "weak" carcinogen, [3H]-1,2,3,4-dibenzanthracene (3,4-DBA), to whole nuclei and nuclear subfractions was also determined. The concentration, specific activity, and time of treatment were identical with those used for MC. The level of binding of [3H]-1,2,5,6-dibenzanthracene was approximately 3-fold greater in whole nuclei on a per mass DNA basis than in those of either the MC or the 3,4-DBA. The binding of MC and 3,4-DBA to whole nuclei was approximately equal. As with MC, the [3H]-1,2,5,6-dibenzanthracene demonstrated a peak of high specific activity binding to the slower-sedimenting fraction of chromatin while the 3,4-DBA displayed considerably less binding to this fraction.


Antitumor and immunosuppressive effects of mycophenolic acid derivatives. Thirth-three derivatives of mycophenolic acid obtained by modifying phenolic hydroxyl and/or carboxyl groups were examined for antitumor and immunosuppressive activities. Some compounds showed more potent antitumor activities to L1210 leukemia and Ehrlich solid tumor than did mycophenolic acid. Most of these suppressed the production of antibody against sheep red blood cells in mice as strongly as did the parent substance. Correlation between antitumor and immunosuppressive activities was generally observed. However, a few compounds possessed a potent antitumor activity with less or no immunosuppressive activity.


Reduction of ifosfamide toxicity using dose fractionation. Ifosfamide was given in i.v. doses of 600 to 1200 mg/sq m/day for 5 days to 32 cancer patients, refractory to prior therapy, in an attempt to investigate the possibility of reducing toxicity by dose fractionation. Microscopic hematuria occurred in 14% and gross hematuria in only 10% of the patient trials. Azotemia did not occur in any patient on this study. Reversible myelosuppression was comparable to that found by other investigators. Other side effects such as nausea and mental confusion occurred infrequently. Ifosfamide produced antitumor effect in 7 of 27 evaluable patients. This study indicates that the renal and bladder toxicity of ifosfamide can be substantially reduced if the drug is administered in i.v. infusions of 1 to 2 hr daily for 5 days.


Delayed and progressive myocardial lesions after adriamycin administration in the rabbit. A cardiomyopathy similar to that observed in cancer patients after prolonged chemotherapy with adriamycin can be produced in the rabbit, characterized by mitochondrial and myofilamentous degeneration with noninflammatory myolysis and connective tissue repair. The progressive and delayed myocardial lesions produced in rabbits receiving increasing total doses of adriamycin were studied after 23, 35, 43, and 77 days of continuous treatment and in posttreatment periods up to 150 to 180 days after the last administration of adriamycin. Total adriamycin doses that produced increased serum enzyme, myocardial sodium, and calcium levels but that failed to elicit serious myocardial morphological lesions during the period of drug administration resulted in a delayed cardiomyopathy leaving focal areas of fibrosis and progressive lesions that became more severe after discontinuation of drug administration. These findings suggest that the myocardial effects of adriamycin are cumulative and potentially nonreversible.


Comparative pharmacologic study in vitro and in vivo with cyclophosphamide (NSC-26271), cyclophosphamide metabolites, and plain nitrogen mustard compounds. This paper deals with the problem of the relative selectivity of the antitumor effect of cyclophosphamide (CP). CP and its metabolites are pharmacologically characterized by determining their chemical and biological activities in vitro and their pharmacotherapeutic properties in vivo. Of particular importance is the specificity of the cytotoxic activity (cytostatic units/mumol) in vitro and the margin of safety (therapeutic index) in vivo. The pharmacologic data reveal: a. Of the various metabolites of CP only 4-hydroxycyclophosphamide, the primary activation product, exerts a highly specific cytotoxic activity in vitro and has a wide margin of safety in vivo. b. The decisive step in toxication is the formation of the alkylating N,N-bis(2-chloroethyl)phosphorodiamidic acid after acrolein has been split off.


The problem of oncostatic specificity of cyclophosphamide (NSC-26271): Studies on reactions that control the alkylating and cytotoxic activity. The relatively high oncostatic specificity of cyclophosphamide (CP) in vivo is shown to be due to the cytotoxic specificity of 4-hydroxycyclophosphamide (4-hydroxy-CP), the first product of metabolic activation of CP in the liver. This specificity can be evaluated not only in vivo by measuring the therapeutic index, but also in vitro by determining its cytotoxicity against Yoshida ascites tumor cells. Evidence is given that 4-hydroxy-CP is not an alkylating agent itself, but attains this property only by release of an alkylating N,N-(2-chloroethyl)phosphorodiamic acid moiety and acrolein. The energetic source for this rate-limiting toxication results from the resonance stabilization of the released acrolein. Reactions at the cryptoaldehyde group of 4-hydroxy-CP, which reduce or prevent the resonance stabilization of the 3-carbon unit to be released, lead to a deactivation of the primary metabolite of CP thus reducing or even preventing toxication, and hence influencing both the alkylating and cytotoxic activities of the molecule. Accordingly, it could be demonstrated by the reaction of 4-hydroxy-CP with thiols yielding 4-(S-R)-mercapto CP derivatives that the toxication of 4-hydroxy-CP can be controlled under physiologic conditions of pH and temperature. In the case of free protein sulfhydryl groups, this reaction also leads to fixation onto a macromolecule of the CP metabolite. On the basis of these peculiar reactivities of the oxazaphosphorine ring of 4-hydroxy-CP and of the partial reaction kinetics involved during toxication or deactivation, the significance of these findings to the problem of CP specificity is discussed.


Aldophosphamide: synthesis, characterization, and comparison with "Hohorst's aldophosphamide". Synthesis of aldophosphamide was attempted by many standard aldehyde-forming reactions under a variety of conditions. Trace amounts of aldophosphamide were produced in several of the reactions, as judged by mass-spectral evidence. A stabilized derivative, aldophosphamide semicarbazone, was prepared and characterized by infrared and proton magnetic resonance, and mass spectral analysis) of "Hohorst's aldophosphamide" failed to reveal any evidence for the existence of an aldehyde moiety, and thin-layer chromatographic comparison with the synthetic analogs diethylaldophosphamide and diethylhomoaldophosphamide indicated radical differences in migration rates between those of the analogs and that of "Hohorst's aldophosphamide." It is suggested that "Hohorst's aldophosphamide" is perhaps a diastereoisomer of 4-hydroxycyclophosphamide or an aldehyde hydrate or hemiacetal with 1,3-propanediol.


Studies on the in vivo formation of acrolein: 3-hydroxy-propylmercapturic acid as an index of cyclophosphamide (NSC-26271) activation. 3-Hydroxypropylmercapturic acid (MCA) has been quantitatively determined in the urine of rats given cyclophosphamide (CP), related antineoplastic agents, allyl alcohol, or acrolein, with a simple procedure involving the use of an amino-acid analyzer. Male rats (300-400 g) injected with CP (50mg [179.1 mumols]/kg of body weight) excreted 16.7 mumols of MCA/kg in their 24-hour urine. Equivalent amounts of isophosphamide produced 9.0; triphosphamide, 16.1; ASTA-5607, 7.2; ASTA-5122, 4.1; and cytoxyl alcohol, 0.4mumols of MCA/kg. From allyl alcohol and acrolein, 26.3 and 19.7 mumols of MCA/kg were obtained respectively. MCA values were directly proportional to drug dose levels. Since acrolein and phosphorodiamidic acid mustard are the toxic decomposition products of aldophosphamide, and acrolein conjugation appears to be the first step for MCA formation values for MCA would reflect active CP levels. The in vitro interaction of acrolein with glutathione, other sulfhydryl compounds, and a few amino acids at concentrations of 0.15 mumols/ml was also studied. The decrease of acrolein's main absorption peak at 209 nm was used to follow its reaction rate. The faster interactions observed were with the sulfhydryl compounds, where a 50% decrease of absorption in interactions with glutathione and cysteine (at pH 7.4 and 23 degrees C) took place in 111 and 30 seconds respectively. Incubation of these adducts at 37 degrees C and 100 degrees C generated acrolein with a maximum recovery yield of 83% at 100 degrees C. Five patients given 1 g of CP iv excreted 6.4-50 mumols of MCA in their urine in 6 hours.


Cyclophosphamide (NSC-26271)-related phosphoramide mustards- recent advances and historical perspective. The antitumor activity of three of the most phosphoramide mustards, NSC-69947, NSC-72505, and NSC-72510, was compared with that of phosphoramide mustard (NSC-69945), the apparent active metabolite of cyclophosphamide (CP), against the L1210, P388, B16, and Lewis lung tumor systems. This comparison did not reveal any significant differences in the patterns of inhibitory activity predictive of significant advantages in the clinic of any of these compounds over CP or NSC-69945. Attempts to prepare aldophosphamide, the key intermediate in CP metabolism, by oxidation of hydroxyphosphamide under the Sarett reaction conditions lead primarily to 4-ketocyclophosphamide. Under milder conditions the product isolated appears to be the elusive aldophosphamide on the basis of positive alkylating and aldehyde tests, Rf and infrared data, and the formation and characterization of a semicarbazone. The possibility that this product is an equilibrium mixture with the tautomeric 4-hydroxycyclophosphamide has not been as yet defintely ruled out. Sarett oxidation of homohydroxyphosphamide straightforwardly gives the stable analog, homoaldophosphamide. Biologic testing of this putative aldophosphamide in direct comparison with homoaldophosphamide, CP, and NSC-69945 reveals that aldophosphamide is a potent antitumor agent indistinguishable in activity from CP and NSC-69945, whereas homoaldophosphamide is inactive. These results provide confirmatory evidence for the postulated role of aldophosphamide as an intermediate in CP metabolism and suggest, furthermore, that aldophosphamide itself is not active in vivo, requiring transformation to NSC-69945 via beta-elimination of acrolein to exert its antitumor effects. An historical account of the development of the phosphoramide mustard field is also given.


Cyclic phosphoramide mustard (NSC-69945) derivatives of amino acids and peptides. The possibilities offered by a combination of the "latency" and "carrier" principles were studied by joining the phosphoramide mustard group with amino acids. The structures of the resulting derivatives are reminiscent of cyclophosphamide, but they provide feasibility for stereoisomeric modifications and for incorporation into synthetic peptides. Modlel peptides with a different ring system containing cyclic diesters of the N,N-bis(2-chloroethyl)phosphoramidic acid were also constructed. The relative intensities of antitumor activity, toxicity, and antitumor spectrum can be influenced by these molecular manipulations, indicating that the pharmacokinetics, and probably even the enzymatic activation, are markedly dependent upon the overall physiochemical properties and stereochemistry of the molecule. Another approach  has been to use larger molecular weight materials as carriers either of the phosphoramide mustard group or the cyclic amino acid derivatives. Both noncovalent and covalent linkages were tried; based on stability properties, conjugates must be regarded as more adequate potential candidates for lysosomotropism.


Deactivation of cyclophosphamide (NSC-26271) metabolites by sulfhydryl compounds. The reaction of 4-hydroxycyclophosphamide (4-hydroxy-CP) with sulfhydryl (SH) compounds was studied, and although the cytotoxic activity was lost a significant stabilization of the alkylating capacity was observed at the same time. We were able to show that the reaction of 4-hydroxy-CP with thiols lead to an equilibrium between the reaction product and the starting substrates. On the basis of this equilibrium the increased stabilization of the alkylating capacity of the 4-hydroxy-CP derivatives by raising the SH concentration can be explained. The different degrees of stabilization depending on the structure of the thiol results from different equilibria. the effect on the toxification reaction resulting from this equilibrium, in connection with the tautomeric equilibrium between 4-hydroxy-CP and aldophosphamide, is discussed.


Stereochemistry, metabolism, and antitumor activity of 4-hydroperoxyisophosphamide (NSC-227114) and its stereoisomer. An acid-catalyzed isomerization of 4-hydroperoxyisophosphamide, a synthetic compound with potential antitumor activity, produced a new crystalline epimer having an inverted stereochemistry at the phosphorus atom. The stereochemistry of these epimeric hydroperoxides is discussed on the basis of their chemical and nuclear magnetic resonance properties. The epimers show essentially the same metabolic behavior. This behavior is, however, different from that of isophosphamide (IP) which is metabolized in part via pathways ineffective for its activation. The newly isolated epimer shows slightly higher activity against some kinds of experimental tumors than the original hydroperoxide, suggesting that the inverted stereochemistry of the alkylating group at the phosphorus atom is effective in promoting the antitumor activity of the activated species of IP.


Importance of pharmacokinetic studies on cyclophosphamide (NSC-26271) in understanding its cytotoxic effect. Pharmacokinetic studies on cyclophosphamide (CP) and its alkylating metabolites produced by hepatic biotransformation have been performed in vivo in animals and in vitro in the perfused liver. CP levels were determined by a gas-chromatographic method combined with mass-spectrometry, and production of alkylating metabolites was assayed by the 4-(4-nitrobenzyl)pyridine reaction for alkylating compounds. Differenes in serum drug levels between normal rats and rats bearing Walker 256 carcinosarcoma were observed in vivo and were confirmed by the liver-perfusion technique. Pharmacokinetic parameters and enzyme kinetic data both in normal and in tumor-bearing animals will be presented. The disappearance of CP and the corresponding formation of CP metabolites was significantly modified when (a) CP was given after previous treatment with a compound which alters its biotransformation (ie, phenobarbital, an inducer of microsomal metabolism), (b) CP was given after previous treatment with CP (which inhibits microsomal metablism), or (c) CP was given with competitive substrates of aldehyde oxidase or dehydrogenase (glyceraldehyde, chloral hydrate, and disulfiram). Results obtained in animals or in the perfused liver will be discussed. The significance of this modified CP metabolism in influencing its cytotoxic effect will be discussed and correlations between drug levels and activity will be presented.


Approaches to the pharmacokinetics of cyclophosphamide (NSC 26271): Quantitation of metabolites. Recent evidence has implicated phosphoramide mustard as a highly active metabolite of cyclophosphamide (CP). This compound has been shown to be produced from CP both in vitro and in vivo. To further study the role of this active metabolite we have devised methods to quantitate the compound in human serum and urine using gas chromatography-chemical ionization mass spectrometry. Tetradeuterated phosphoramide mustard has been synthesized and is used as an internal standard. CP and nor-nitrogen mustard have also been quantitated in human samples using analogous tetradeuterated internal standards.


Sparing effect of cyclophosphamide (NSC-26271) pretreatment on animals lethally treated with gamma-irradiation. Cyclophosphamide (CP) can protect mice from gamma-irradiation-induced lethality, the timing of the dose of CP in relation to the irradiation being the critical factor. The most sparing effect is achieved when CP is injected 3 days before irradiation. The protection is mediated by enhanced hemopoietic recovery seen in animals given the most favorable combination ofCP and gamma-irradiation. This rapid recovery cannot be explained by the reduced sensitivity of hemopoietic stem cells after CP treatment to subsequent irradiation although survival of a few radioresistant stem cells cannot be ruled out. Stimulus to repopulate by nonspecific depletion of the bone marrow is not the explanation either, since CP itself appears to play an important role in this effect. The role of the spleen is not critical in this phenomenon and attempts have failed to demonstrate the presence of a humoral factor which can restore bone marrow depleted by gamma-irradiation in the serum of animals treated with CP. CP given 3 days before irradiation appears to result in a purely additive effect against malignant tissue in mice, providing a useful differential kill on tumor tissue at the same time that it provides the maximum protection of normal tissue.


Identification and pharmacokinetics of cyclophosphamide (NSC-26271) metabolites in vivo. The existence of the cyclophosphamide (CP) metabolites, 4-hydroxycyclophosphamide and aldophosphamide, in vivo after application of CP to mice and rats has been demonstrated. The metabolite pattern in the serum and urine of rats at different times after CP application was determined. For this purpose, thin-layer chromatograpic procedure was developed which allows separation of the metabolites without decomposition. Because considerable amounts of radioactivity are bound to serum proteins after 3H-CP application, protein binding studies with CP and CP metabolites were carried out. It was found that 4-hydroxycyclophosphamide is bound to thiol groups of bovine serum albumin yielding a rather stable product which could be isolated by column chromatography on Sephadex.


Permeation of cyclophosphamide (NSC-26271) metabolites into tumor cells. The permeation kinetics of cyclophosphamide and its metabolites were studied with Ehrlich ascites tumor cells, murine L1210 leukemia cells, and mouse L929 fibroblasts at 1 degrees C. In contrast to carboxyphosphamide and nor-nitrogen mustard, an equipartition of cyclophosphamide and 4-hydroperoxycyclophosphamide was observed in these cells. First experiments have been done to study the efflux of cyclophosphamide and 4-hydroperoxycyclophosphamide after incubation until saturation with Ehrlich ascites tumor cells. Cyclophosphamide could be washed out completely, whereas, 4-hydroperoxyclophosphamide shows an apparent retention in the cell at 37 degrees C.


Studies on 4-hydroperoxycyclophosphamide (NSC-181815): a simple preparation method and its application for the synthesis of a new class of "activated" sulfur-containing cyclophosphamide (NSC-26271) derivatives. 4-Hydroperoxycyclophosphamide was obtained in approximately 20% yield by ozonization of cyclophosphamide in acetone/water at 0 degrees C. It was reduced to 4-hydroxycyclophosphamide, which was treated with several mercaptans to yield compounds of the type 4-(S-R)-mercapto-cyclophosphamide. In the solid state these compounds are stable at room temperature; in aqueous solution they are hydrolyzed to 4-hydroxycyclophosphamide or its tautomer, aldophosphamide. One of the 4-(S-R)-mercapto-cyclophosphamide compounds was tested biologically in vitro against Yoshida ascites tumor cells and showed the same cytotoxic activity as 4-hydroxycyclophosphamide.


Studies on the metabolism of isopnosphamide (NSC-109724) in man. A method is described for the quantitative determination of cyclophosphamide, isophosphamide, and trophosphamide and their stable alkylating metabolites on thin-layer chromatography plates with the aid of 4-pyridinealdehydr-2-benzothiazolylhydrazone (PBH). The method requires only 0.01-0.02 ml of urine from patients treated with high doses of cyclophosphamide of isophosphamide, but a careful standarization is required by using reference substances. In the urine of patients treated with isophosphamide, the 4-carboxy derivative and two different dechloroethyl derivatives resulting from side-chain oxidation are the predominant metabolites determined, next to isophosphamide. Side-chain oxidation may be prevalent. Determination of isophosphamide and its stable alkylating metabolites in the urine of 25 patients shows that the ratio of unchanged isophosphamide to its metabolites varies widely. The mean value of the total amount of PBH-reactive compounds exceeded 50% of the given dose, whereas less than 1% was found to be converted to urine metabolites which release acrolein.


Studies on the human pharmacokinetics of isophosphamide (NSC-109724). The pharmacokinetics of isophosphamide (IP) in man has been studied using 14C-labeled drug and differential extraction of unchanged drug from metabolites by CHCl3 extraction of plasma and urine. The plasma decay of IP is biphasic with a terminal half-life of 15.2 hours which is twice that reported for cyclophosphamide (CP). The fraction of drug metabolized is 49% for IP compared to 88% reported for CP. This is consistent with the six-times larger pseudometabolic pharmacokinetic rate constant reported for CP (0.26 hr-1) than found by us for IP (0.04 hr-1). The renal clearance for IP (21.3 ml/min) is two times that of CP (10.7 ml/min). In addition to an analysis of the kinetics of disposition and elimination of unchanged IP, an analysis of the pharmacokinetics of total metabolite of IP in plasma and urine was made. A nonlinear pharmacokinetic model which includes a term for biotransformation according to Michaelis and Menten revealed that the total metabolites do not diffuse from a small plasma space of 2.1 liters but are excreted directly via the urine. In contrast to the biexponential plasma decay of single, high-dose IP (5 g/m2), multiple-dose IP (2.4 g/m2/day X 3) shows a monoexponential decay with a half-life of 6.9 hours, a pseudometabolic rate constant of 0.08 hour-1, and a renal clearance of 18.7 ml/minute. The fraction of drug metabolized (79.7%) calculated from the pharmacokinetic parameters agrees with the fraction of drug recovered in urine as total metabolites (72.8%). This is distinctly different from high-dose IP (48.6%) but similar to the fraction of drug metabolized as reported for CP (88.0%). This suggests that the pharmacokinetic transfer constant for IP metabolism is dose dependent, an observation that may be useful in developing new IP regimens.


Some studies on the distribution and effects of cyclophosphamide (NSC-26271) in normal and neoplastic tissue. The gross uptake and intracellula distribution of 32P-cyclophosphamide has been studied in normal and neoplastic rat and mouse tissues. Prolonged alkylation of DNA and RNA occurs over periods of 24 hours in the mouse and 48 hours in the rat, and it is suggested that this may involve slow release of active metabolites from a drug-macromolecule complex. The survival of cells in pulmonary and sc tumors after cyclophosphamide treatment has been studied in situ using a biochemical assay, and the influence of environmental factors at the tumor site has been assessed. The pattern of drug distribution has been studied in large sc tumors, and in one tumor line the drug concentration achieved has been related to estimated cell survival.


Distinctive effects of mechlorethamine (NSC-762), cyclophosphamide (NSC-26271), and BCNU (NSC-409962) on transcription in Ehrlich cells. Comparisons have been made between mechlorethamine, cyclophosphamide (CP), and BCNU as to the effects of chemotherapeutic drug levels on the transcription of RNA in vivo in Ehrlich cells. Mechlorethamine causes a dose-dependent depression in the synthesis of large Hn RNA transcripts in the nucleus and in the amount of large polysomal mRNA in the cytoplasm without apparent disturbance of polyadenylation and transport. Because of compensating increases in smaller Hn RNA and mRNA chains there is no inhibition of total RNA synthesis. CP seems to accelerate total Hn RNA and mRNA formation but inhibits polyadenylation. CP also produces an excess of short chains with only a minor depression of the synthesis of longer transcripts and messages. BCNU inhibits both RNA synthesis and polyadenylation but without disturbance of the size distribution of the polynucleotide chains. The results with mechlorethamine, in particular, are not easily explained on the basis of premature chain termination at the sites of DNA alkylation.


Aspects of the teratology of cyclophosphamide (NSC-26271). Wistar-derived rat embryos were most sensitive to maternally administered cyclophosphamide on Days 9.5 and 10.5 of gestation and to chlorambucil on Day 9.5 of gestation. The spectrum of malformations and the dose-response behavior of both drugs were similar. Chlorambucil was three times more potent than cyclophosphamide on an mg/kg basis. The drugs showed additive behavior when administered in combination. The teratogenic effects of the individual or combined drugs were reduced by protective doses of cysteine or glutathione. Studies with analogs of cyclophosphamide have suggested that the whole molecule is involved in teratogenesis.


The use of deuterated analogs in qualitative and quantitative investigations of the metabolism of cyclophosphamide (NSC-26271). Ring-deuterated analogs of cyclophosphamide (CP) (4-d2, 5-d, 4,6-d4, and 4,5,6-d6 derivatives) have been used to study the influence of deuterium substitution on the rates of metabolic pathways involving oxidation at C-4, and on the rate of elimination of acrolein from aldophosphamide. The magnitude of the deuterium isotope effect (kH/kD) associated with appropriate C-deuteration has been related to antitumor activity against the ADJ/PC6 murine plasma cell tumor. Isotope effects of 2.2 and 1.8 respectively, for the formation of 4-ketocyclophosphamide (4-keto-CP) and carboxyphosphamide, caused little or no change in antitumor activity (4-d2 and 4,6-d4 analogs compared with CP), but an isotope effect of about 5.3 for the beta-elimination pathway, consequent on 5,5-dideuteration, was paralleled by a marked drop in potency (7-13-fold increase in ED90) of 5,5-dideuterated analogs compared with that of CP. Analogs tetradeuterated in the bis(2-chloroethyl)amino function were used to quantitate CP and 4-keto-CP in human plasma and urine using stable-isotope dilution and direct-insertion electron impact mass spectrometry. The negative optical rotation of CP recovered from human urine after administration of the racemlc drug gave evidence for stereoselectivity in the metabolism.


Studies on the disposition of cyclophosphamide (NSC-26271) in tumor-bearing mice by means of gas chromatography-chemical ionization-mass fragmentography. The disposition of cyclophosphamide (CP) in mice bearing Lewis lung tumor has been studied by chemical ionization-mass fragmentography using isophosphamide as the internal standard. The drugs were analyzed as their N-trifluoroacetyl derivatives in order to increase sensitivity and to avoid column adsorption and decomposition phenomena. The minimum detectable amount was 500 pg/injection. Linearity was found up to microgram amounts of the substance without any interference of endogenous compounds.


Qualitative and quantitative investigations of cyclophosphamide (NSC-26271), cyclophosphamide metabolites, and related compounds by field-desorption mass spectrometry. The potential of field-desorption mass spectrometry (FD-MS) coupled with photoplate detection for qualitative and quantitative studies on cyclophosphamide (CP) and its metabolites is reviewed. The characteristic features of this ionization mode are minimal fragmentation of the molecular ion coupled with the capacity to produce such ions from thermally unstable or nonvolatile samples. Since CP is itself extensively fragmented in the electron-impact ionization mode, and since it possesses both thermally unstable (eg, 4-hydroxycyclophosphamide, carboxyphosphamide) as well as ionic and hence nonvolatile metabolites (eg, phosphorodiamidic acid), the FD technique has potential value for the detection and quantitation of the drug and its metabolites in underviatized mixtures. Some of the factors which influence the reproducibility of FD spectra are enumerated. In order to improve the FD technique for quantitative results a device for emission-controlled FD was constructed and its use is described. Three different modes of operation are discussed using the FD spectra of CP conjugates typical for each. Preliminary investigations into the use of the d6 analog of CP for quantitation of the drug by FD-MS are also reported.


Characterization of cyclophosphamide (NSC-26271) metabolites and related derivatives by field-desorption and electron-impact mass spectrometry. Several important metabolites of cyclophosphamide (CP), such as 4-hydroxycyclophosphamide and phosphoramide mustard, some 4-alkyl(aryl)thio derivatives have been investigated by field-desorption and electron-impact mass spectrometry. The structural identification of synthetic compounds and of derivatives, isolated by thin-layer chromatography in vitro, was possible since complementary information can be obtained using the two ionization techniques. Whereas only the field-desorption mass spectra showed more abundant molecular ions, the electron-impact technique revealed a characteristic fragmentation pattern for most of the compounds studied. Examples are given which demonstrate the existence of stereoisomers of CP derivatives using thin-layer chromatography and field-desorption mass spectrometry. The results indicate that field-desorption mass spectrometry is also a particularly appropriate method for the characterization of unstable CP metabolites in vivo.


X-ray crystallographic studies of cyclophosphamide (NSC-26271) derivatives. Molecular structures of 4-hydroperoxycyclophosphamide (4-hydroperoxy-CP) and of two stereoisomers of 4-hydroperoxyisophosphamide (4-hydroperoxy-IP) have been determined by X-ray crystallography. All compounds, synthesized by Dr. A. Takamizawa by ozonolysis of appropriate open-chain precursors, demonstrate in vivo and in vitro cytotoxicity. The structure results show that for 4-hydroperoxy-CP and both isomers of 4-hydroperoxy-IP the 4-hydroperoxy group is situated axial to the ring. In 4-hydroperoxy-CP and the isomer of 4-hydroperoxy-IP which is the major product of the ozonolysis, the phosphoryl oxygen is equatorial to the ring and trans to the C4-oxygen. A scheme is presented to account for the interconversion of the two 4-hydroperoxy-IP configurations. Since a stereoisomer with a hydroperoxy or hydroxy group equatorial has not been isolated from the ozonolysis synthesis or from the Fenton oxidation of cyclophosphamide (CP), the configuration with C4-oxygen axial which is found in 4-hydroperoxy-CP and both isomers of 4-hydroperoxy-I is probably more stable and may be the configuration of 4-hydroxy compounds formed by the in vivo oxidation of CP, isophosphamide, and trophosphamide (TP). The molecular structure of TP has also been elucidated. Comparison of the conformations of the three chloroethyl alkylating groups of this molecule with those of the two chloroethyl groups of other CP derivatives indicates that these groups are highly flexible and do not assume one favored conformation.


The influence of dietary protein deficiency on haemopoietic cells in the mouse. These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1-4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs--although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18%) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstructive role during the refeeding process.


A technique for determining the proportion of the clonogenic cells in S phase in ENT6 cell cultures and tumors. The survival of cultured EMT6 cells was examined after treatment with hydroxyurea (HU) or high specific activity tritiated thymidine (3H-TdR). The concentrations of the agents, duration of exposure to the agents, and post-exposure treatment of the cultures were found to influence the cell survival; the effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and by 3H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of 3H-TdR. Under these conditions, either 3H-TdR or HU could be used to determine the proportion of the clonogenic cells in S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or 3H-TdR, using the conditions found optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and as the proportion labeled by 3H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogeneic tumor cells in S phase. Incubation of tumor cell suspensions  with 3H-TdR in vitro proved difficult to perform and the results were relatively unreliable because of severe problems with reutilization of 3H-TdR during the incubation for colony formation.


Alterations in 3H-thymidine incorporation into DNA induced by methyl CCNU (1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea) in normal and tumorous tissues in vivo. Methyl CCNU produces a suppression of tritiated thymidine (3H-TdR) incorporation into DNA in vivo in normal bone marrow and gastrointestinal tissues which is different in magnitude and duration from that seen in L1210 ascites tumor in the same animals. This suppression and recovery pattern is not seen in animals bearing L1210 ascites tumor resistant to MeCCNU. Where a different pattern of recovery is seen between normal host target tissues and tumor, the pattern can be exploited to increase the cure rate of animals bearing advanced L1210 ascites tumor with properly spaced second doses of MeCCNU. Additional information on the potential toxicity of second doses of MeCCNU can be predicted from knowledge of the time of recovery of DNA synthesis in the normal host target tissues.


The migration of lymphocytes across specialized vascular endothelium. II. The contrasting consequences of treating lymphocytes with tryspin or neuraminidase. Lymphocytes were exposed in vitro to either trypsin or neuraminidase. The ability of the treated cells to migrate into tissues were measured (a) by i.v. injection into intact recipients and (b) by vascular perfusion through an isolated lymph-node preparation. The localization of trypsinized cells in the lymph-nodes of recipients was deficient when compared to untreated lymphocytes and there was a surplus of trypsinized cells in blood. Trypsinized cells migrated into the isolated nodes in reduced numbers. By contrast, neuraminidase treated lymphocytes were markedly deficient in the blood of recipients early after injection; their localization in the spleen and lymph-nodes was also deficient but they were in surplus in the liver. Moreover they migrated into the isolated nodes in slightly increased numbers. By 24 hr after injection the perturbed localization pattern produced by either enzyme was partly restored to normal. In conclusion, tryspin interfered with the capacity of lymphocytes to migrate into lymph-nodes but neuraminidase did not; the latter promoted the hepatic sequestration of cells and the reduced localization in the blood and tissues was a consequence of this. The hypothesis that lymphocytes adhere to specialized endothelia in lymph-nodes because of specific glycoside sequences on their surface lacks experimental support.


The effects of colcemid on mouse bone marrow. Following Colcemid administration, mitoses accumulate preferentially in the subendosteal region of the bone marrow of the mouse. This finding suggests that the most rapidly proliferating cells are localized to the subendosteal region, and complements previous radioautographic studies which have demonstrated a corresponding labelling gradient in the marrow. Quantitative estimates of cell cycle time by the stathmokinetic method were precluded by the presence of significant Colcemid induced interphase cell loss. Colcemid also affected cell differentiation in the marrow. Following Colcemid administration there was a fall in mature granulocytes in the marrow, and a concommitant rise in marrow megakaryocytes.


Properties of Go cells: variations in the proliferative response following isoprenaline. The proliferative behaviour induced in the acinar cells of the rat submaxillary gland in response to isoprenaline has been used to examine the transit time of cells from a quiescent (Go) state into the S phase. Cumulative 3H-TdR labelling index curves were constructed to determine the mean time interval (Gis time) between stimulation with isoprenaline and entry into the S phase. Data were collected for the proliferative wave induced by three sequential injections of isoprenaline, and the effects of varying the interval between the second and third injections of isoprenaline, and of changing the dose of the drug, were examined. Intervals of 28, 52 and 76 hr between isoprenaline injections resulted in mean Gis times of 16-2, 20-9 and 25-6 hr respectively. It was concluded that the Gis time depended on the recent history of cells with respect to stimulation, but not division. The results are considered in terms of two models, in one of which the time to leave Go is variable, whilst in the other the cells leave Go immediately the stimulus is applied.


The 'transition probability model' and the regulation of proliferation of human diploid cell cultures during aging. Genealogies derived from time-lapse cinemicrophotographic studies of aging human diploid cell cultures were analysed in terms of the 'transition probability' model. It was found that the distribution of intermitotic times obtained from middle passage cells deviated only slightly from that predicted by the model. In contrast, the plot for late passage cultures did not fit the predicted pattern and appeared to be composed of multiple curves. These changes are discussed in reference to cellular senescence as expressed by normal human diploid cells in vitro.


Morphology and distribution of supraependymal cells in the third ventricle of the albino rat. The supraependymal cells (SEC) are a normal component of the wall of the cerebral ventricles. In the hypothalamic area of the third ventricle they are restricted, in healthy animals, to the ependymal projection of the hypophyseotropic area. Here the SEC show great polymorphism. In addition to bipolar, multipolar and stellate or spider-like cells, transitional forms between these types can be seen. Their perikarya and processes can either remain at some distance from the ependyma or be in close contact with it. The processes may protrude between the ependymal cells or show surface differentiations that resemble the thin cytoplasmic folds of the mesenchymal wandering cells. Considering this and the variations in the number of cells, for example during the ovarian cycle, the SEC can be interpreted as mesenchymal cells, probably related to microglial cells of the subependymal layer. It is suggested that the SEC have a phagocytotic function and may be involved in the normal renewal of the ependyma. A definitive explanation for the restriction of the SEC to the hypophyseotropic area as well as the elucidation of their function remain to be found.


Ultrastructure of the compound eye of the haploid male beetle, Xyleborus ferrugineus. The compound eye of male (haploid) Xyleborus ferrugineus beetles was examined with scanning and transmission electron microscopy. The eye externally consists of ca. 19 to 33 facets. Each ommatidium is composed of a thickly biconvex lenslet with about 50 electron dense and rare layers, but at the junction area between two lenslets there are only about 35 to 37 layers that can be distinguished. A very short (3.4-4.0 mum) acone type crystalline cone is located directly beneath the lenslet. Each ommatidium is surrounded by pigment cells, and pigment granules also appear throughout the cytoplasm of the retinular cells. Some pigment granules are even present below the basement membrane. there are 8 retinular cells. The rhabdomeres of 2 centrally situated photoreceptor cells fuse into a rhabdom which is enveloped by the rhabdomeres of 6 peripheral retinular cells. The rhabdomeres of the 6 peripheral retinular cells join laterally to form a rhabdomeric ring around the central rhabdom. No tracheation was observed among the retinular cells. Virus-like particles are evident near the nucleus in each Semper cell of the crystalline cone.


Transport of material between the nucleus, the chromatoid body and the Golgi complex in the early spermatids of the rat. The movement and transport of material between intranuclear dense particles, the chromatoid body and the Golgi complex have been studied in early spermatids of the rat. The analyses involved observation of living accurately identified cells, time-lapse cinemicrography and electron microscopy. The chromatoid body establishes transient contacts with intranuclear material during early spermiogenesis. The chromatoid body also makes contacts with the Golgi complex. It is suggested that the chromatoid body receives material from the nucleus during the postmeiotic period and particites in the early formation of the acrosomic system.


Retinohypothalamic pathway in the duck (Anas platyrhynchos). Retinohypothalamic connections were studied in the duck after unilateral optic nerve transection using both light and electron microscopic techniques. Degenerated endings of optic fibers were found only in a circumscribed part of the anterior hypothalamic area, i.e. the ventral region of the contralateral suprachiasmatic nucleus. Images of degenerating boutons were observed in frozen sections (method according to Johnstone-Bowsher), and their presence confirmed by electron microscopic examination. These degenerating boutons make synaptic contacts with dendrites or dendritic spines of neurons of the suprachiasmatic nucleus. In the same material, the decussation of the optic chiasma was studied with the light microscope. Uncrossed retinal fibers were found in the marginal optic tract, the basal optic root and occasionally also inthe isthmo-optic tract.


Effect of calcium depletion and subsequent repletion on parathyroids, parafollicular (C) cells and bone in the growing pig. The ultrastructure of the chief cells of the parathroid gland and thyroid parafollicular (C) cells and the morphology of bone in calcium depletion and subsequent repletion were examined in young growing pigs. A low calcium diet resulted in osteopenia, increased removal of the cartilaginous core, osteoclasia and osteocytic osteolysis. Subsequent repletion quickly returned bone to normal. In pigs fed the low calcium diet, there was a marked depletion of secretory granules but a striking increase in the number of microtubules in chief cells. Increasing the calcium content of the diet to normal quickly returned the ultrastructural appearance of chief cells to apparent normal. In the initial response to calcium repletion, chief cells exhibited large number of lysosomes and occasionally prominent  paracrystalloid bodies. Electron microscopic examination of parafollicular (C) cells of the thyroid gland failed to reveal differences in ultrastructure between test and control pigs. These findings support the view that bone resorption following calcium deficiency may be the result of a secondary hyperparathyroidism rather than of calcium deficiency per se.


Fine structure of neurons of the arcuate nucleus and median eminence of the hypothalamus of the golden hamster following immobilization. The fine structure of arcuate neurons of the arcuate nucleus, the ependymal tanycytes and the contact zone of the median eminence was examined following immobilization, an acute stress which significantly activated the hypothalamo-pituitary-adrenal (HPA) axis. Arcuate neurons of immobilized adult male hamsters displayed morphological indications of heightened activity; the number of lysosomes and dense core vesicles (80-120nm) was increased. A markedly greater number of dense core vesicles was present in axon terminals of the contact zone of the mid-central median eminence and the ventral proximal stalk. Tanycytes of the median eminence exhibited an augmented number of electron dense bodies in both perikarya and end processes. These results indicate that the arcuate neurons, the axons of the contact zone, and the ependymal tanycytes of the hamster medial basal hypothalamus (MBH) may be involved in the response to immobilization.