Evaluation of fast neutron teletherapy for advanced carcinomas of the major salivary glands. This review analyzes the local control rate of fast neutron beam teletherapy for malignant salivary gland carcinomas compared to conventional photon irradiation at the same institution. Thirty patients with malignant parotid or submaxillary gland tumors were treated with either cobalt-60 (19 patients) or neutrons +/- photons (11 patients). The 19 photon-treated patients were historical controls. No advantage is found for neutrons in patients with tumors less than 3 cm where 100% local control was achieved with either modality. In patients with tumor masses 3-6 cm in size, neutrons demonstrated a possible advantage with a local control rate of 100% (followup, 7-18 months) compared to a control rate of 33% with conventional photon irradiation. Acute treatment-related morbidity was slightly greater in neutron-treated patients, but no long-term complications have been observed to date.


MACC chemotherapy for adenocarcinoma and epidermoid carcinoma of the lung: low response rate in a Cooperative Group Study. Eastern Cooperative Oncology Group. The MACC (methotrexate, adriamycin, cyclophosphamide, CCNU) regimen was administered to 43 patients with advanced epidermoid and adenocarcinoma of the lung. Only 5 patients (12%), all of whom were ambulatory responded with partial remissions. Median time to progression for the 5 responders was 20 weeks from start of treatment. Median survival was 15.5 weeks for patients with epidermoid cancer and 14.4 weeks for those with adenocarcinoma. Hematologic toxicity was severe, with 2 treatment-related deaths during profound myelosuppression. White blood counts below 2000/microliter were reported in 47%, and below 1,000/microliter in 26%. Since the activity of this regimen, given as it was in full doses, is not superior to that achieved with standard doses of single agents which are less toxic, further employment of the MACC regimen is not recommended, either for advanced disease or as a surgical adjuvant.


Malignant peritoneal mesothelioma following radiotherapy for seminoma of the testis. The difficulties encountered in establishing a diagnosis of a malignant peritoneal mesothelioma are emphasized in a patient who developed the lesion 16 years after radiation therapy for a seminoma of the testis. Historically, histologically, electron microscopically, and by microincineration, there was no evidence of asbestos exposure. We believe that present lesion may be a consequence of prior radiation therapy.


A fatal case of inappropriate ADH secretion induced by cyclophosphamide therapy. Cyclophosphamide is used extensively to treat malignancies. A 5-year-old boy with stage IV neuroblastoma is described who developed a fatal syndrome of inappropriate antidiuretic hormone (ADH) secretion after high dose cyclophosphamide therapy.


Neutrophil function in lymphoreticular malignancy. Neutrophil chemotaxis, phagocytosis, and metabolic stimulation were studied in 25 patients with untreated lymphoreticular malignancies, 8 patients with untreated carcinomas, and 8 patients undergoing therapy for lymphoreticular tumors. Phagocytic activity and metabolic stimulation during phagocytosis were comparable to controls in all patient groups. Slight, but significant, differences were found in chemotactic responsiveness of untreated patient groups when compared to controls. In almost all instances, chemotactic responsiveness reverted to normal in most patients studied after therapy for the malignant process. In patients with intact immunologic function these minor differences in chemotactic responsiveness are probably not sufficient to predispose to infection. However, this defect could be additive, and when present in combination with impaired immunity may contribute to compromised host defense.


Histopathologic changes of nasal mucosa in nickel workers: a pilot study. Histologic examination of standard biopsy specimens from nasal mucosa were performed on 98 former and present employees at a nickel refinery and 61 controls. Histologic characteristics of the epithelium were graded blindly according to a point score table. The histologic scores were compared to certain epidemiologic data, such as the degree and duration of nickel exposure, duration of industrial work without nickel exposure, age, and smoking habits. High histologic scores were correlated to high nickel exposure, to the duration of this exposure, to age, and to industrial work not involved with nickel. One person with nasal carcinoma was detected in the nickel-exposed group. During the investigation period, three additional nasal carcinomas in former nickel workers were discovered and later included in the study. Furthermore, 18 cases of epithelial dysplasia were found in the nickelexposed group, but none among the controls. The cases with nasal carcinomas were or had been working with processes that gave an estimated high nickel dust exposure. All the individuals with epithelial dysplasia were nickel process workers.


Noninvasive precursor lesions of adenocarcinoma and mixed adenosquamous carcinoma of the cervix uteri. It is postulated that squamous cell carcinoma, adenocarcinoma, and mixed adenosquamous cell carcinoma of the uterine cervix all have a common cell or origin, the subcolumnar reserve cell. The relative frequency with which the various types of carcinoma in situ are seen is in part explained by the ubiquitous nature of squamous metaplasia in the region of the transformation zone in women of reproductive age. It is suggested that squamous metaplasia is the soil on which most squamous carcinomas of the cervix evolve. The relatively low frequency with which adenocarcinoma and adenosquamous carcinoma in situ are encountered may also be dependent on their less accessible location in the endocervix.


Synergistic effect of diethylstilbestrol on the mutagenicity of 2-acetylaminofluorene and N-hydroxy-acetylaminofluorene in the Salmonella assay system. Salmonella typhimurium, TA-1538, was used to investigate the mutagenic potential of N-2-acetylaminofluorene (2-AAF), N-hydroxy-N-2-acetylaminofluorene (N-OH-2-AAF) and diethylstilbestrol (DES) individual and in combination. In the presence of an induced or uninduced rat liver metabolizing system (S-9), the histidine requiring strain of bacteria was reverted to prototrophy by the aromatic amines but not by the synthetic estrogen. However, when DES was combined with 2-AAF or N-OH-I-AAF in the presence of the induced S-9 fraction, the number of revertant colonies was increased 2- to 4-fold above the levels obtained with the aromatic amines alone. The synergistic effect of DES, a non-mutagen, on the mutagenicity of these aromatic amines was observed only when a 3-methylcholanthrene induced rat liver S-9 fraction was used as the source of mammalian enzymes. When uninduced mouse or rat liver S-9 fractions were used in this test system, an inhibitory effect rather than an enhancing effect was observed.


Reactions of N-methyl-N-nitrosobenzylamine and related substrates with enzyme-containing cell fractions isolated from various organs of rats and mice. Incubation of the following N-nitroso compounds with microsomes from mouse liver and an NADPH-generating system formed the products stated: benzaldehyde from N-methyl-N-nitrosobenzylamine (MNBA), acetone and formaldehyde from N-methyl-N-nitroso-(alpha-phenyl)-ethylamine (MNPE) and formaldehyde from N-methyl-N-nitroso-(2-phenyl)-isopropylamine (MNPI). Enzymes isolated from mouse liver showed more activity towards MNBA by comparison with rat liver preparations. Microsomes from the organs of female rats showed decreasing activity in the following sequence: esophagus greater than forestomach greater than liver and for female mice: liver greater than forestomach.


Epidermal prostaglandins after topical application of a tumor promotor. The topical application of the promoter, 12-O-tetradecanoyl-13-acetate, on the shaved backs of mice was attended by a striking increase in the PGE concentration within epidermis as early as 1 h later. This increase peaked at 24 h but the prostaglandin value remained elevated for at least 48 h. PGF, on the other hand, showed only a modest increase in epidermis with a maximum at 12 h. By 48 h, the PGF concentration returned to normal.


The morphologic and biologic patterns of chemically induced pancreatic adenocarcinoma in Syrian golden hamsters after homologous transplantation. A pancreatic adenocarcinoma induced by N-nitrosobis(2-oxopropyl)amine in the Syrian golden hamster was successfully transplanted to a homologous host by subcutaneous inoculation through 10 successive passages. The rate of 'tumor take' increased progressively with each generation from 60% to 100%, and the latency period after inoculation was reduced simultaneously from 6 weeks to 1 week in the second and following passages. The tumors grew rapidly, ulcerated the overlying skin, and metastasized to the regional lymph nodes and lungs. The animals usually died with multiple lung metastases between the 5th and 20th weeks. All transplanted tumors and their metastases retained the pattern of the original, well-differentiated adenocarcinomas.


Mutagenicity of N-nitroso pyridinol carbamate with the Salmonella/mammalian microsome test. Pyridinol carbamate was nitrosated to the N,N1-dinitroso derivative and the structure was proved by spectroscopic methods, including chemical ionization mass spectroscopy. By the Ames test, the dinitroso derivative showed a significant dose-dependent mutagenic response with Salmonella typhimurium strains TA 1535 and TA 100; the response became more pronounced in the presence of microsomes. As the dosage of N-nitroso pyridinol carbamate increased, the number of revertant colonies also increased. Pyridinol carbamate was not mutagenic.


Differential excision from DNA of the C-8 deoxyguanosine reaction products of N-hydroxy-2-aminofluorene and N-acetoxy-N-acetyl-2-aminofluorene by endonuclease S1 from Aspergillus oryzae. Calf thymus DNA was modified in vitro with [G-3H]N-hydroxy-2-aminofluorene and [G-3H]N-acetoxy-N-acetyl-2-aminofluorene and the nuclease S1 digestion was studied under identical conditions. The ratios of the maximum reaction rate (V) and the Michaelis constant (Km), V/Km, indicate that 2-aminofluorene(AF)-modified DNA is hydrolyzed 3 times more slowly than N-acetyl-2-aminofluorene(AAF)-modified DNA under similar reaction conditions. The AF-modified DNA was slightly more susceptible to partial digestion by nuclease S1 than unmodified control DNA. These results suggest that the local regions of denaturation induced by AF substitution are smaller than those associated with AAF modification.


Aryl hydrocarbon hydroxylase induction in mitogen-stimulated lymphocytes by benzanthracene or cigarette tars adsorbed to asbestos fibers. Human mitogen-stimulated lymphocytes, cultured in the presence of amosite asbestos (AS), demonstrated a slight increase in aryl hydrocarbon hydroxylase (AHH) activity compared with non-induced (control) cultures (P = 0.005). A much greater increase in enzyme activity occurred following addition of the inducers benzanthracene (BA) or cigarette tars (CT) to cell cultures (P less than 0.001 in both instances). Significant enzyme induction also occurred when AS fibers were first preincubated with CT or BA, washed with acetone, then added to lymphocyte cultures (P less than 0.003 in all instances). This increase in AHH activity was not as great, however, as the induction observed when BA or CT was added to cell cultures. No further increase in enzyme activity was noted when AS and CT or AS and BA were simultaneously added to culture lymphocytes (P greater than 0.070 in all instances). The results demonstrate that polycyclic aromatic hydrocarbons (PAH), such as BA and other components of CT, are adsorbed and transported by amosite AS particles. These AS-PAH complexes are capable of inducing AHH in cultured human lymphocytes.


Effects of temperature and time on mutagen formation in pan-fried hamburger. Mutagenic activity generated in hamburger during pan-frying is dependent upon both temperature and time, with temperature appearing to be the more important variable. Uniformly prepared frozen hamburger pattie (115 g; 19% fat) were fried under carefully controlled conditions at 143 degrees C, 191 degrees C and 210 degrees C. Mutagenic activity assayed with the Ames test was not detected in uncooked hamburger, and in hamburger fried at 143 degrees C mutagenic activity remained low at all times studied (4--20 min). In contrast, frying at 191 degrees C or 210 degrees C for up to 10 min resulted in the generation of considerably higher levels of mutagenic activity. Mutagenic activity in fried hamburgers sold at selected restaurants ranged from very low to moderately high. Evidence is also presented for mutagenic inhibitory activity in uncooked and fried hamburger. Mutagenic inhibitory activity decreased mutagenesis mediated by liver S-9 from normal rats but not from Aroclor 1254-treated rats.


High incidence of rat mammary carcinoma by oral administration of ethyl methanesulphonate. Ethyl methanesulphonate (EMS), one of the alkylating agents, is known to be a potent mutagen. We tested EMS for its carcinogenicity in female rats by oral administration. EMS was dissolved in drinking tap water at a concentration of 10(-3) M and was given for the first 12 weeks. The subcutaneous tumors were noticed as early as 16 weeks after initiating the experiment. At the 32nd week, all the surviving rats produced tumors; the majority were multiple tumors in the neck, axillar and inguinal areas corresponding to bilateral mammary glands. Histologically, the prevailing feature of the tumors was infiltrating medullary adenocarcinoma consistent with carcinoma of mammary duct origin. Neither regional lymph node involvement nor distant metastases were shown, but intraductal spread of carcinoma was a marked finding during the 32-week period.


Enhancement of experimental photocarcinogenesis by topical retinoic acid. Topical application of retinoic acid (RA) solutions greatly enhanced the response of hairless mouse skin to a moderate dose of simulated sunlight. Tumors appeared much earlier, and in much greater numbers, in animals treated daily with 1 or 10 micrograms of RA in methanol immediately after 2 h exposure to a xenon arc filtered through 2 mm of Schott WG 320 glass (approximately equivalent in human erythema effectiveness to 5 min of mid-summer noon solar exposure in northern mid-latitudes), compared to mice treated with light and methanol only. The higher amount of RA, in combination with light, produced moderate epidermal hyperplasia and some scaling and transient erythema, but no gross ulceration or inflammation of skin. The lower amount of RA, though about equally effective in carcinogenesis, produced minimal epidermal hyperplasia compared to the ultraviolet radiation + methanol control.


Further studies of the effect of pepstatin on ascites accumulation in tumor-bearing mice. Studies were done of the effect of pepstatin on ascites accumulation in mice bearing MM46, Ehrlich, CCM, SN36, L1210, and NTF ascites tumors. When pepstatin was injected subcutaneously at 80 mg/kg body wt before ascites accumulation, it inhibited the accumulation in all strains of the tumors tested. In MM46, CCM and NTF tumor strains there was also a decrease in the tumor cell numbers following pepstatin treatment. Kinetic studies on ascites accumulation with tumor strain MM46 demonstrated that even when pepstatin was injected after ascites accumulation it reduced the ascites volume. A dose-dependent effect was observed in this tumor strain when pepstatin was injected both before and after ascites accumulation. The results confirm previous studies of pepstatin's ability to retard ascites in L1210 and P-815Y ascites tumors and also broaden the concept of the mechanisms by which petstatin may be acting.


Comparison of the skin tumor initiating activity of 3-methylcholanthrene and 3,11-dimethylcholanthrene in mice. The abilities of 3-methylcholanthrene (3-MC) and 3,11-dimethylcholanthrene (3,11-DMC) to initiate skin tumors in Sencar mice were determined by using a 2-stage system of tumorigenesis. 3,11-DMC was found to have very weak skin tumor initiating activity when compared to the potent activity of 3-MC. The only difference between 3-MC and 3,11-DMC is the substitution of a methyl group in position 11 which is part of the 'K-region' or the 'peri' position. From these results, we suggest that an unhindered peri position adjacent to an angular benzene ring is necessary for carcinogenic activity of 3-MC.


Effect of methylprednisolone on the cell kinetic response of C3H/HeJ mammary tumors to cyclophosphamide and adriamycin. The effect of methylprednisolone (MP) on the cell kinetic response to cyclophosphamide (CP) and Adriamycin (ADR) in C3H/HeJ spontaneous mammary tumors and hematopoietic tissue was investigated. The [3H]deoxythymidine labelingg index, the primer-dependent DNA polymerase labeling index (an estimate of tumor growth fraction), and the mitotic index were determined at various intervals after treatment. Treatment consisted of CP (200 mg/kg) on Day 0 plus ADR (2 mg/kg) on Day 1 with or without MP every 12 hr for 9 doses beginning on Day 2. In tumors treated with CP and ADR alone, changes in the kinetic parameters suggested proliferative recovery between Days 3 and 4 which coincided with bone marrow recovery. In tumors treated with CP, ADR, and MP, although the timing of the hematopoietic recovery was not affected by MP, the overshoot of the [3H]deoxythymidine labelin index on Days 3 and 4 was abolished. Proliferative recovery in the tumor was delayed until after cessation of MP treatments. Cell kinetic changes in the tumor after CP, ADR, and MP were used to design effective sequential chemotherapy which obviated the hematopoietic toxicity associated with sequential therapy designed from cell kinetic changes after CP and ADR alone.


Influence of immune status on the metastasis of three murine fibrosarcomas of different immunogenicities. Three fibrosarcomas of different immunogenicities were tested for their ability to form spontaneous and experimentally induced metastases in normal, sham-suppressed, immunosuppressed, and immunologically restored syngeneic mice. Immunosuppression, achieved by adult thymectomy and sublethal X-irradiation (450 R), affected experimental metastasis of the three tumors in different ways. Upon i.v. injection, the highly immunogenic fibrosarcoma formed more pulmonary tumor colonies in immunosuppressed mice than in normal, sham-suppressed (sham thymectomy and 450 R), or immunologically reconstituted animals (thymectomy, X-irradiation, plus 10(7) normal syngeneic lymphocytes given i.v.). A fibrosarcoma of intermediate immunogenicity also formed more pulmonary metastases in immunosuppressed recipients, but this increase could not be reversed by reconstitution with 10(7) lymphocytes. In contrast, the least immunogenic tumor formed fewer pulmonary tumor colonies in immunosuppressed mice than in normal, sham-suppressed, or immunologically reconstituted mice. We conclude that the role of the immune system in experimental cancer metastasis varies for different tumors and that tumor immunogenicity is an important factor in the relationship between host immunity and tumor dissemination.


Synergistic action of 5-aza-2'-deoxycytidine and 3-deazauridine on L1210 leukemic cells and EMT6 tumor cells. The biochemical and biological effects of the combination of 5-aza-2'-deoxycytidine (5-aza-dCyd) and 3-deazauridine (3-DU) on L1210 leukemic cells and EMT6 tumor cells were investigated. The cytotoxic action of 5-aza-dCyd and 3-DU on both L1210 and EMT6 cells in vitro was synergistic when these agents were used in combination. The combination of 5-aza-dCyd and 3-DU produced a greater inhibition of in vitro growth of L1210 and EMT6 cells than did either agent alone. The in vivo antineoplastic activity of this combination was synergistic with respect to the increased survival time of BALB/c x DBA/2 F1 mice with L1210 leukemia. 3-DU, an agent that reduces the intracellular pool size of cytosine nucleotides, stimulated the incorporation of [3H]-5-aza-dCyd into DNA of both L1210 and EMT6 cells, suggesting that the synergistic action of this combination is related to the increased incorporation of 5-aza-dCyd in the presence of 3-DU.


Suppression of tumor-associated hyperfibrinogenemia and free fatty acidemia with p-phenoxybenzalbutyrate (clofibrate). Previous work in our laboratory has indicated that free fatty acids stimulate synthesis of fibrinogen by the liver. The effect of the hypolipidemic agent clofibrate on hyperfibrinogenemia associated with tumors was evaluated by monitoring clofibrate-induced changes in plasma fibrinogen concentration and biosynthesis of the protein in Buffalo rats implanted with a localized, nonmetastasizing neoplasm derived from a tumorigenic hepatoma cell line (HTC4). In tumor-bearing animals not treated with clofibrate, cancer growth was associated with elevated rates of fibrinogen synthesis and a doubling of plasma fibrinogen concentrations. Plasma free fatty acid concentrations and serum free fatty acid/albumin molar ratios were also increased in tumor-bearing rats. Treatment with clofibrate in doses which normalized the plasma free fatty acid/albumin ratio also prevented the tumor-associated rise in plasma fibrinogen. Rates of fibrinogen synthesis were lowered significantly in clofibrate-treated animals. Tumor growth was not affected by clofibrate. These results indicate that hyperfibrinogenemia associated with nonmetastasizing tumors may reflect changes in lipid metabolism which are neutralized by clofibrate. Thus, treatment with clofibrate or other hypolipidemic agents should be evaluated in cancer patients with elevated plasma fibrinogen levels and their attendant complications.


Galactosyl transferase activity in human transitional cell carcinoma lines and in benign and neoplastic human bladder epithelium. Cultured cells of human transitional cell carcinoma line MGH-U1, in suspension, were assayed for galactosyl transferase by measurement of the transfer of [3H]galactose from uridine diphosphate:[3H]galactose to desialylated ovine submaxillary mucin. The assay was optimized with respect to time and to protein, uridine disphosphate:galactose, desialyated ovine submaxillary mucin, and Triton X-100 concentrations. This assay was then applied to fresh specimens of benign, inflamed, and neoplastic bladder epithelium from 33 patients who under went cold-cup biopsies at cytoscopy. Transitional cell carcinoma specimens gave values in the range of 24.7 to 184.8 cpm [3H]galactose transferred per microgram protein per hr [72.0 +/- 44.7 (S.D.); n = 25]; normal and inflamed specimens ranged from 0.8 to 46.1 cpm/microgram protein per hr [8.3 +/- 8.4 (S.D.); n = 35]. By using a known method of cell rupture, cell ghosts, representing cell-surface membranes, were isolated both from the cultured cell line and from two biopsy specimens of transitional cell carcinoma. Although a complete enzymatic and electron microscopic analysis was not undertaken, the coincidence of an enzyme marker with the cell ghost fraction containing the elevated galactosyl transferase made it appear probable that this enzyme is located in the cell surface.


Effect of surgical removal on the growth and kinetics of residual tumor. Findings from this study using a transplantable C3H mammary tumor failed to indicate interaction relative to growth parameters between two foci present in the same host. Whether they were growing alone or in the presence of a second focus, tumor growth rates were similar until the combined mass of multiple tumors approached that which was incompatible with survival. Only then was a difference in growth observed. Cytokinetic parameters, i.e., labeling index, primer-dependent DNA polymerase index or growth fraction, DNA synthesis time, tumor doubling time, and cell cycle time, were also similar whether tumors grew alone or in the presence of a second focus. Following removal of a tumor, changes were observed within 24 hr in the kinetics of the residual focus. There was an increase in labeling index (duration approximately equal to 10 days) and primer-dependent DNA polymerase index with a decrease in the tumor doubling time. Minimal change was noted in DNA synthesis time and cell cycle time. The kinetic changes observed were reflected in a measureable increase in tumor size approximately equal to a week following tumor removal. Absence of an alteration in DNA synthesis time and cell cycle time indicates that the increase in tumor growth was probably due to a conversion of noncycling cells in G0 phase into proliferation. Relationship of the findings to the use of adjuvant chemotherapy is considered.


Activity of thymidine as a chemotherapeutic agent against human tumor xenografts in nude mice. The chemotherapeutic activity of thymidine (dThd) was tested against four human tumor xenografts growing in nude mice, including a melanoma, an oat cell carcinoma of the lung, a colon carcinoma, and a breast carcinoma. Tumor-bearing mice were given an infusion of dThd (1 g/kg/day) s.c. for 72 hr each week for three weeks. Tumor growth in the treated mice was compared to that in randomized concurrent control mice infused with media alone. A significant effect was found only for the melanoma, and it was cytostatic rather than cytotoxic. Even when melanomas of very small initial volume were treated, there were no complete regressions, and tumor growth resumed when dThd treatment was stopped. In culture, sustained dThd concentrations of greater than 3.2 mM were required to cause death of the melanoma cells; in the mice the dThd level during infusion ranged from 1 to 5 mM. This exposure to dThd, although failing to produce a tumor response, did produce significant toxicity in the nude mice in the form of myelosuppression and leukopenia. Flow cytometric analysis of marrow cells during the dThd infusion showed an accumulation of cells in S phase, but proliferation was not completely halted since cells with G2-M content of DNA were present in the marrow even after 72 hr of dThd exposure. This study failed to demonstrate a therapeutically useful effect of dThd on these tumors.


Phase I clinical and pharmacological study of 4'-(9-acridinylamino)-methanesulfon-m-anisidide using an intermittent biweekly schedule. 4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA, NSC 249992), an acridine derivative, was given to 28 patients with solid tumors and one patient with Hodgkin's disease in a Phase I clinical trial. The dose schedule used was a single dose given every 14 days for three doses. The amount given ranged from 10 to 120 mg/sq m/dose. Dose-limiting toxicity was moderate to severe leukopenia which occurred at and above 70 mg/sq m. Thrombocytopenia was infrequent and did not require transfusion. Nonhematological side effects were mild and included nausea, vomiting, local irritation, and fever. Antineoplastic activity was noted in liposarcoma, adenocarcinoma of unknown primary origin, and squamous carcinoma of unknown primary origin (one patient each). Pharmacokinetics studies were done in 19 patients. Total m-AMSA and free m-AMSA concentrations showed a biphasic distribution with an initial rapid phase of t1/2 = 10 to 15 min for both, and a second slow phase of t1/2 = 8 to 9 hr for total m-AMSA and 3 hr for free m-AMSA. Phase II studies with m-AMSA, in hematological cancers are warranted, since its most consistent effect is on leukocytes. The recommended dosages for solid-tumor Phase II studies are 70 mg/sq m for good-risk patients and 50 mg/sq m for poor-risk patients, given as a single dose every other week, or 120 mg/sq m for poor-risk patients for the single-dose every-3-week schedule.


Establishment of photoaffinity label derivatives of fluorene as probes in studies of chemical carcinogenesis in mammalian cell culture. Several azido fluorenes, photosensitive analogs of the established carcinogen 2-acetylaminofluorene, have been synthesized to serve as probes in chemical carcinogenesis and mutagenesis studies. Unlike 2-acetylaminofluorene, these compounds do not require metabolic activation. However, alkylation to critical targets is achieved through the generation of nitrenes by photolysis at 360 nm in situ. We have found that the bifunctional azidofluorenes 2,5-diazidofluorene and 2,7-diazidofluorene were more toxic to and more transforming of the mouse embryo C3H 10T1/2 clone 8 cells when photolyzed in situ than were the monofunctional azides 2-azidofluorene and 7-bromo-2-azidofluorene. When the drugs were photolyzed in phosphate-buffered saline (pH 7.4) and then added to the cells these preirradiated derivatives were not transforming but were slightly toxic. However, there is not relationship between the number of photosensitive azido groups and the cytotoxicities of the preirradiated derivatives as measured by the plating efficiency method. The transformation experiments were performed under conditions in which photolysis was carried out for as little as 15 sec. This is significant because the major events that lead to oncogenesis apparently can occur within this narrow time channel, thereby making the drugs excellent probes in studies in chemical carcinogenesis. Although near ultraviolet light alone may transform the cells (8 to 16 min of irradiation), under the conditions studied ultraviolet light was neither toxic nor transforming.


Isolation and characterization of canine venereal tumor-associated inhibitory and blocking factors. Spontaneous regression of the canine venereal tumor is associated with the production of a serum factor which inhibits in vitro tumor colony-forming units in agar. Logarithmic or persistent tumor growth, on the other hand, is characterized by a serum factor which protects cells against in vitro inhibition (blocking factor). These factors have been characterized by immunochemical methods. Whole regressor and blocking sera were fractionated by Sephadex G-200 filtration and immunoabsorption with rabbit antiserum specific for canine immunoglobulin G2a. Fractions were characterized by immunoelectrophoresis, radial immunodiffusion, and disc gel electrophoresis. In vitro inhibitory and blocking activity of the whole serum was accounted for by the purified immunoglobulin G2a. Blocking activity was also found in protein eluted from logarithmically growing tumors. Preparative polyacrylamide electrophoresis revealed five major fractions with blocking activity only in the immunoglobulin G fraction. Tumor eluates and immunoglobulin G isolated from serially removed tumors demonstrated with the clinical course of the tumor. Using ultrafiltration and sodium dodecyl sulfate electrophoresis of tumor-associated immunoglobulin G at low pH, it was not possible to identify an antigen complexed to the blocking antibody.


Effect of phorbol ester tumor promoters on the expression of melanogenesis in B-16 melanoma cells. Cells of the C3 clone of B-16 melanoma synthesize melanin only at confluence after which they senesce and can no longer be passaged. Addition to the cultures of 10(-8)--10(-7) M 12-O-tetradecanoylphorbol-13-acetate (TPA) shortly after plating delayed by about 2 days the onset of melanogenesis. TPA did not, however, affect the growth of the cells or the time at which they reached confluence. The ability of a series of phorbol esters to delay melanogenesis correlated with their tumor-promoting activity on mouse skin. The optimum time for addition of TPA was within the first 24 hr after plating; the inhibitory effect decreased when TPA was added at later points. alpha-melanocyte-stimulating hormone (5 x 10(-7) M) added to B-16 cultures 24 hr after plating slowed the growth of the cells and caused them to differentiate when still subconfluent. TPA also inhibited this alpha-melanocyte-stimulating hormone-induced melanogenesis. These results suggest that TPA inhibits a very early stage in a stepwise process that leads to the differentiation of these cultures. For reasons that are not apparent, the cells eventually escape from this inhibition. The B-16 melanoma cell culture system may be useful for studying the mechanism by which TPA and related tumor promoters affect cellular differentiation.


Formation of carbonyl chloride in carbon tetrachloride metabolism by rat liver in vitro. In order to identify intermediates of CCl4 metabolism, whole, suitably fortified rat liver homogenates were incubated with 14CCl4 in the presence and absence of "pools" of unlabeled suspected intermediates. In the presence of NADH or NADPH, incorporation of radioactivity was rapid and substantial in CO2, lipid, protein, and the acid-soluble fraction. It was not influenced by the presence of large pools of unlabeled chloroform or formate, thus excluding these substances as obligatory intermediates. However, when incubated with L-cysteine, radioactivity incorporation in the acid-soluble fraction was almost doubled, and about one-third of the radioactivity of this fraction was identified as 2-oxothiazolidine 4-carboxylic acid. This substance is formed chemically by condensation of cysteine with carbonyl chloride and has been identified previously by others as a product of chloroform metabolism by liver microsomes in the presence of L-cysteine. Based on current knowledge of CCl4 metabolism, the following aerobic pathway is envisioned: microsomal cleavage to Cl- and .CCl3 and oxidation of the latter to the unstable intermediate, Cl3COH, which loses HCl to yield COCl2. COCl2 is likely to be the major source of CO2 from CCl4 but is probably not the intermediate that binds to lipid and protein. The addition of glutathione had no effect on CCl4 metabolism in rat liver homogenate, suggesting that glutathione S-transferases, which catalyze other dehalogenation reactions, do not play a role in CCl4 metabolism.


Utilization of 2'-deoxy-6-thioguanosine 5'-triphosphate in DNA synthesis in vitro by DNA polymerase alpha from calf thymus. Chemically synthesized beta-2'-deoxy-6-thioguanosine 5'-triphosphate, a putative active form of beta-2'-deoxy-6-thioguanosine, was used efficiently as a substrate for DNA synthesis catalyzed by DNA polymerase alpha from calf thymus. The deoxythioguanylate was incorporated into DNA by replacing deoxyguanylate and supported the further elongation of DNA chains on activated calf thymus DNA. In contrast, DNA polymerase beta used beta-2'-deoxy-6-thioguanosine 5'-triphosphate at a much lower rate. The reaction product of DNA polymerase alpha, i.e., 6-thioguanine-containing DNA, adsorbed specifically to the organomercurial agarose column, and showed a peak of UV absorption at 342 nm, which is characteristic of thioguanine.


Influence of factors derived from EMT6 tumors and from bone marrow of tumor-bearing mice on tumor and bone marrow stem cell kinetics. Untreated EMT6 tumors in BALB/c mice were used to assess the regulatory mechanisms of tumor growth in these animals. This tumor can be quantitated for clonogenic cells by in vitro techniques, and the hydroxyurea suicide method makes it possible to determine the kinetic status of the clonogenic cells. The untreated EMT6 tumor does not seem to have internal humoral regulatory mechanisms explaining tumor growth kinetics. However, the exponentially growing EMT6 experimental tumor releases a factor capable of stimulating quiescent splenic colony-forming units into cycle. This is also true of bone marrow taken from tumor-bearing mice. This study was made possible using an in vivo-in vitro technique which separates the effector cells from the responder cells by a Millipore filter floating on the culture medium. The relationship between tumor growth and normal hematopoietic tissue of the tumor-bearing animal is discussed.


Sensitivity to chemotherapeutic and immunomodulating agents of two mouse lymphomas and of a macrophage tumor. The effect of treatment with 15 chemotherapeutic and 10 immunomodulating agents on the growth of T-cell lymphoma EL-4, macrophage tumor J774, and B-cell lymphoma 70Z/2 of the mouse has been studied using the prolongation of median survival time of tumor-bearing hosts as an index of therapeutic effectiveness. The survival time of mice bearing 70Z/2 was prolonged more than 100% by single-agent therapy with actinomycin D, cyclophosphamide, 6-mercaptopurine, mitomycin C, and vinblastine; a similar response of J774 was produced by therapy with Adriamycin, cyclophosphamide, or 6-mercaptopurine. No chemotherapeutic agent prolonged the median survival time of mice bearing EL-4 by 100% or more. Of the immunomodulating agents, mycobacterial preparations (Bacillus Calmette-Guérin or interphase material), Corynebacterium parvum, and polyinosinic-polycytidylic acid moderately prolonged the survival time of mice bearing J774 or 70Z/2; the EL-4 lymphoma was refractory to all 10 immunomodulating agents.


Effect of the duration of retinyl acetate feeding on inhibition of 1-methyl-1-nitrosourea-induced mammary carcinogenesis in the rat. The effect of the duration of retinoid treatment on the inhibition of 1-methyl-1-nitrosourea-induced mammary carcinogenesis was studied. Female Sprague-Dawley rats were given i.v. injections of 50 mg 1-methyl-1-nitrosourea per kg body weight at both 50 and 57 days of age. Feeding of a placebo diet or diet supplemented with 323 mg retinyl acetate per kg diet (retinoid treatment) was initiated at 10 days after the first carcinogen injection. Retinoid treatment was either continued or discontinued after 60 days postcarcinogen, and the study was terminated at 182 days postcarcinogen. Retinoid treatment between 10 and 60 days postcarcinogen prolonged the cancer latency and reduced the average number of cancers per rat in comparison to that in placebo-treated rats. Continuation or cessation of retinoid treatment in 60-day tumor-bearing rats had no effect on the time of appearance of additional cancers. In 60-day tumor-free rats, continuation of retinoid treatment prolonged cancer latency in comparison to either 60-day tumor-free rats changed to placebo or rats continuously treated with placebo. The cessation of retinoid treatment in 60-day tumor-free rats resulted in a rapid increase in the appearance of cancers; at the termination of the study, the average number of cancers per rat was similar to that of animals fed only the placebo. The data indicated that some rats are more responsive to the retinoid than are others. Retinoid treatment apparently prevented the progression of early neoplastic lesions, and a continuous daily intake of the retinoid appears necessary to sustain the chemopreventive effect under the experimental conditions imposed.


Hormonally induced tumors of the reproductive system of parabiosed male rats. Parabiosis of intact male rats to castrated males or oophorectomized females for a period of approximately 20 months resulted in three interstitial cell tumors of the testis. When unilateral nephrectomy was added to the parabiotic procedure in ten pairs, eight interstitial cell tumors of the testis and four adenocarcinomas of the prostate occurred in the target male parabionts. These changes were preceded by elevations in luteinizing and follicle-stimulating hormone levels in the serum of the castrates and high levels of testosterone and, to a lesser degree, of androstenedion in the target partners developing the tumors.


Effect of tetrahydrouridine on the action of 1-beta-D-arabinofuranosylcytosine in synchronized cultures of normal rat kidney cells. Tetrahydrouridine (THU) is a reduced pyrimidine nucleoside and has been found to be a potent cytidine deaminase inhibitor. We have examined the effect of THU on the action of 1-beta-D-arabinofuranosylcytosine (ara-C) on DNA synthesis and its cytotoxic effect on normal rat kidney (NRK) cells. The results indicated that THU, when present alone, has no effect on the DNA synthesis or viability of the NRK cells. The addition of THU can, however, further increase the cytotoxic effect of sublethal doses of ara-C and can enhance its ability to inhibit DNA synthesis. Studies on the duration of exposure of the NRK cells to ara-C and on the effect of ara-C on synchronized cultures of NRK cells also support the earlier reports that ara C, through its active compound 1-beta-D-arabinofuranosylcytosine 5'-triphosphate, is capable of blocking cells from progression from G1 into S phase of the cell cycle. Furthermore, we have shown that this inhibition is reversible after the drug has been removed. The addition of THU in the presence of noninhibiting levels of ara-C can also result in the blocking of cells from progression into S phase of the cell cycle. Similar to the effect of ara-C, this inhibition is also reversible. These studies suggest that the addition of THU enhances the cytotoxicity of sublethal doses of ara-C and its ability to inhibit DNA synthesis.


Physicochemical differences between glucocorticoid-binding components from the corticoid-sensitive and -resistant strains of mouse lymphoma P1798. The physicochemical properties of nuclear and cytosolic glucocorticoid-binding components from corticoid-sensitive (CS) and corticoid-resistant (CR) mouse lymphoma P1798 cells have been compared. Nuclei or cytosol fractions were prepared from lymphocytes that had been labeled at 37 or 4 degrees, respectively, with 30 nM [3H]triamcinolone acetonide ([3H]TA). [3H]TA was extracted with 0.6 M KCl, 10 mM spermidine, or 4.5 mM MgCl2 from CS nuclei and with 0.6 M KCl or 10 mM spermidine from CR nuclei. As reported previously, nuclear-associated [3H]TA in CR cells was resistant to extraction with mM concentrations of MgCl2. Loss of bound steroid during extraction with 0.6 M KCl was minimized by including the chymotrypsin inhibitor, carbobenzoxy-L-phenylalanine, in the extraction buffer. The inhibitor was not required during extraction with spermidine or MgCl2. Nuclear and cytosolic extracts were examined by analytical agarose gel filtration and glycerol density gradient centrifugation under high salt (0.6 M KCl) conditions. The glucocorticoid-binding component in KCl, spermidine, and MgCl2 extracts from CS nuclei was considerably larger and more asymmetrical [Stokes radius, 57 to 59 A; sedimentation coefficient, 3.64 to 3.70S; molecular weight, 90,000 daltons; frictional ratio, 1.8; axial ratio (prolate ellipsoid), 15] than the [3H]TA-macromolecular complex in KCl and spermidine extracts from CR nuclei[Stokes radius, 29 A; sedimentation coefficient, 3.23 to 3.30S; molecular weight, 40,000 daltons; frictional ratio, 1.25; axial ratio (prolate ellipsoid), 5]. Control experiments showed that the smaller size of the glucocorticoid-binding component in CR nuclei was probably not due to cleavage of a larger, CS-like complex during the extraction procedure. The larger size of the CS [3H]TA complex did not appear to result from aggregation of s a smaller species. No difference in physicochemical parameters of the binding component was observed if cells were labeled with [3H]dexamethasone instead of [3H]TA. However, [3H]dexamethasone complexes were less stable than those formed with [3H]TA as indicated by considerable dissociation of [3H]dexamethasone during gel filtration and gradient centrifugation. This may be due to the 3- to 5-fold lower relative binding affinity of [3H]dexamethasone. Analysis of [3H]TA-labeled cytosol by gel filtration and gradient centrifugation revealed the presence of a single binding component with physicochemical properties similar to those of nuclear [3H]TA complexes from the same strain of tumor. These results suggest that previously described differences in extractability of nuclear-associated [3H]TA between the CS and CR strains of mouse lymphoma P1798 and the lack of response of CR P1798 to glucocorticoid administration may be due, at least in part, to the presence of an altered glucocorticoid-binding component in the resistant tumor cells.


Immune-mediated arrest and reversal of established visceral metastases in athymic mice. The metastasizing MDAY-D2 tumor of DBA/2 mice disseminates in BALB/c allogeneic athymic nude (nu/nu) mice in a manner identical to that observed in the syngeneic host. Both the kinetics and organ distribution pattern of metastases from s.c. implants of MDAY-D2 are routinely predictable at any given tumor dose. BALB/c heterozygote (nu/+) litter-mates reject MDAY-D2 grafts on the basis of the multiple minor histocompatibility differences that exist between DBA/2 and BALB/c mice. The in vitro cell-mediated cytotoxic response detected in tumor-bearing BALB/c nu/+ mice is "low grade" (isotope release is approximately 40 to 50% by 24-hr 111-indium-8-hydroxyquinoline assay and approximately 6 to 8% by 6-hr 51Cr assay) and yet correlates directly with tumor rejection. BALB/c nu/nu mice can be protected against MDAY-D2 by previous reconstitution with lymphoid cells from normal or MDAY-D2-sensitized BALB/c nu/+ mice. In addition, surgically documented, established visceral metastases in BALB/c nu/nu mice can be arrested and regressed by the adoptive transfer of MDAY-D2-sensitized BALB/c nu/+ spleen cells. This represents one of the few models where established metastases have been immunotherapeutically regressed. As such, the MDAY-D2 BALB/c nu/nu mouse model offers unique advantages for studying the role of the immune system in regulating the metastatic process.


Advanced stage and early relapse of breast carcinomas associated with high thymidine labeling indices. The thymidine labeling index (TLI) showed a lognormal distribution for 133 primary breast carcinomas with a median of 2.21 and a range of 0.04 to 18.6/100. High TLI's were associated significantly with nuclear anaplasia, pushing tumor border, necrosis, cellular inflammation, and total replacement of lymph nodes involved by tumor, and the data suggested an association of high TLI with large primary tumor and the presence of axillary metastasis. Five patients who initially had local or distant spread all had above median TLI's. They and the 38 patients who developed recurrent tumor during a mean interval of 3.2 years of observation after mastectomy showed a higher geometric mean TLI than those who remained tumor-free (2.93 versus 1.85; p less than 0.05). The rate of early relapse for operable patients with above-median TLI's was significantly greater than that for patients with below-median TLI's (p = 0.0018). High TLI appears to be associated with rapid evolution of breast carcinoma both prior to and following diagnosis and treatment.


Methotrexate-induced changes in the levels of 1-beta-D-arabinofuranosylcytosine triphosphate in L1210 cells. Treatment with methotrexate (MTX) plus 1-beta-D-arabinofuranosylcytosine (ara-C) on Days 1, 4, and 7 after i.p. inoculation of L1210 ascites cells was more effective than treatment with one drug on Days 1, 4, and 7 followed by the second drug on Days 2, 5, and 8. Simultaneous treatment with both drugs was associated with a retention of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) but no increase in the activity of deoxycytidine kinase in L1210 cells, whereas pretreatment with MTX 24 hr before the administration of ara-C was associated with approximately 2-fold increases in the level of ara-CTP and of deoxycytidine kinase in L1210 cells. However, from 5 hr after treatment with ara-C, higher levels of ara-CTP were observed in L1210 cells treated simultaneously with both drugs than in cells from animals pretreated with MTX 24 hr before treatment with ara-C. The superiority of simultaneous treatment over sequential treatment and the synergism between MTX and ara-C, previously reported for this schedule of simultaneous treatment, are attributed in part to the MTX-induced retention of ara-CTP and the increased exposure of L1210 cells to ara-CTP that results from the slower clearance of ara-CTP.


Inhibition of human melanoma colony formation by retinoids. We studied the effects of retinoids on the in vitro survival of melanoma colony-forming cells in biopsies obtained from ten patients with metastatic melanoma. The results indicate that specific retinoids reduce the ability of fresh human melanoma cells to form colonies in soft agar. The retinoids studied had differential effects on the survival of clonogenic melanoma cells, and these effects vary from patient to patient. The data provide support for the clinical trial of selected retinoids in micrometastatic and advanced melanoma.


Isoenzymes of beta-hexosaminidase from normal rat colon and colonic carcinoma. N-Acetyl-beta-D-hexosaminidase isolated from normal rat colon was compared to that obtained from a transplantable rat colonic carcinoma. Levels of total hexosaminidase from purified epithelial cells of normal colon were similar to those from purified malignant cells from the transplantable tumor. Cultured malignant cells had significantly higher levels of activity than did freshly purified tumor cells. Isoelectric focusing of hexosaminidase from normal rat colon indicated approximately equal amounts of A (pI 5.0 ) and B (pI 8.1) isoenzyme activity. The B isoenzyme (normal cell) was more stable to heat inactivation than was the A isoenzyme and had significantly higher activity at low pH's. In contrast, the B isoenzyme from the tumor was relatively unstable to heat and low pH.


Effects of macromomycin on the ultrastructure and biological properties of cultured mammalian cells. Macromomycin is shown to inhibit the biosynthesis of RNA, DNA, and protein in cultured cells of KB, HBL-100, SW-613, MCF-7, and A1Ab. There was no substantial increase in cell numbers in cultures containing macromomycin (5 microgram/ml), but after 24 to 48 hr the cells were two to three times the diameter of control cells with concomitant increase in cell protein. The ultrastructural changes induced by macromomycin in KB cells demonstrate an increase in nuclear size without similar changes in the size of other cytoplasmic subcellular units. It was of interest to note the general proliferation of cellular organelles and the increased occurrence of annulate lamellae followed, after prolonged treatment, by the appearance of larger numbers of lipid droplets and lysosomes; vacuoles developed to a significant extent after the cells detached from the monolayer. A1Ab cells show ultrastructural changes similar to those of KB cells when treated with macromomycin.


Penetration of 3-deazauridine into human brain, intracerebral tumor, and cerebrospinal fluid. The antitumor agent 3-deazauridine (DAU) was administered rapidly to four patients before surgical removal of intracerebral tumor. Tumor, adjacent brain tissue, and temporalis muscle were assayed for DAU by high-pressure liquid chromatography. DAU penetrated comparably into tumor, brain, and muscle; in one patient, tissue concentrations were higher than concurrent plasma concentrations. The active metabolite 3-deazauridine 5'-triphosphate was quantitated in one tumor sample and greatly exceeded its Ki for cytidine 5'-triphosphate synthetase. DAU was also present in autopsy brain specimens from two patients treated shortly antemortem. Cerebrospinal fluid concentrations were 22.1 and 59.0%, respectively, of concurrent plasma concentrations during continuous infusion of DAU in two patients. Cerebrospinal fluid concentration was 3.1 microgram/ml 2 hr after a 30-min infusion of 1.5 g of drug per sq m and fell to 1.9 microgram/ml at 16 hr. Thus, DAU is capable of penetrating into intracerebral tumor, brain, and cerebrospinal fluid and is worthy of investigation in the treatment of intracerebral and meningeal neoplasms.


A selective effect of methylglyoxal-bis(guanylhydrazone) on the synthesis of mitochondrial DNA of cultured L1210 leukemia cells. Methylglyoxal-bis(guanylhydrazone) (MGBG) is a polycationic drug which is useful in the chemotherapy of lymphoid and myeloid proliferative disorders. The drug has recently been shown to produce selective ultrastructural damage to the mitochondria of proliferating cell populations. It is important to understand the molecular basis for this action, since it may be related to the known ability of MGBG to block polyamine biosynthesis. Accordingly, the effect of MGBG treatment on the incorporation of [3H]thymidine into both mitochondrial and nuclear DNA has been examined. Exponentially growing L1210 leukemia cells were prelabeled with [14C]thymidine, treated with MGBG for 1.5 to 16 hr, and then pulse labeled with [3H]-thymidine. Incorporation of [3H]thymidine into mitochondrial DNA was selectively inhibited at 5 hr with concentrations of 1 to 10 microM MGBG. Nuclear DNA, however, was not similarly affected until 8 to 11 hr of drug treatment. Dye-CsCl gradients of mitochondrial DNA indicated that the inhibition of synthesis occurred in replicative forms of circular DNA. Uptake studies excluded the possibility of drug interference with cellular uptake of thymidine. Ultrastructural studies revealed a very close correlation between the dose-response curve for mitochondrial damage and that for MGBG inhibition of mitochondrial DNA synthesis. This correlation suggests a direct cause-and-effect relationship between inhibition of mitochondrial DNA synthesis and ultrastructural damage, but the possibility of both phenomena being related to another action by the drug, such as inhibition of polyamine biosynthesis, or a drug effect on mitochondrial function, must also be considered.


Enhancement of the chromosome-damaging action of ascorbate by transition metals. Freshly prepared ascorbate inhibited mitosis and induced chromosome aberrations in cultured Chinese hamster ovary cells. Cu(II) and Mn(II) (10(-4) or 10(-5) M) enhanced both actions. Fe(II) and Fe(III) (10(-4) or 10(-5) M) reduced or abolished the mitosis-inhibiting action of ascorbate. At 10(-4) M, Fe(II) and Fe(III) strongly enhanced the chromosome-damaging capacity of ascorbate. Up to 100% of all examined metaphase plates had multiple chromosome exchanges or breaks. Since the cytostatic and clastogenic effect of ascorbate of H2O2 to induce chromosome aberrations was examined. H2O2 and a H2O2: Fe(II) mixture (Fenton reagent) induced chromosome breaks and exchanges but to a lesser degree than did ascorbate: Cu(II), Mn(II), Fe(II), or Fe(III) mixtures. Whether the strong chromosome damaging capacity of ascorbate plus transition metals as seen in the in vitro test system poses a health hazard only properly designed in vivo studies can reveal.


Fate of nickel subsulfide during carcinogenesis studied by autoradiography and X-ray powder diffraction. Sarcomas in mice were induced by i.m. and s.c. administration of 63Ni- and 35S-labeled nickel subsulfide (Ni3S2), and the fate of the Ni3S2 was studied in tumors and normal tissues during carcinogenesis. Whole-body autoradiography showed a gradual loss of solubilized 63Ni and 35S radioactivity from the site of injection. There was also a loss of nonsolubilized dust particles which appeared to be phagocytized by reticuloendothelial cells in the liver, spleen, and regional lymph nodes. Microautoradiography showed that the totally dominating radioactivity within both the 63Ni3S2- and the Ni3(35)S2-induced tumors was associated with dust particles. There was no specific or excessive localization of solubilized radioactivity in the tumors or in metastases (when present). Two patterns of localization of dust particles within the tumors were observed: one with particles concentrated in a central part of the tumor and one with the particles present in the periphery of the tumor. X-ray powder diffraction of the insoluble crystalline material in the tumors indicated that a conversion of the alpha Ni3S2 to alpha Ni7S6 and beta NiS had occurred.


Immune elimination of host fibroblasts for the cultivation of human tumors transplanted into nude mice. We report here a useful method for the isolation and cultivation of human tumor cells in vitro from human tumors grown in nude mice. A rabbit was immunized with spleen cells obtained from adult nude mice. The rabbit antiserum in the presence of complement effectively killed cultured cells derived from various mouse tissues, but it was not cytotoxic to cultured cells from human tissues including tumors. When mixed cultures consisting of human tumor cells and nude mouse fibroblasts were treated with the antiserum and complement, the nude mouse fibroblasts were completely removed from the cultures, and the human tumor cells could be propagated without noticeable changes in morphological features. Primary cultures of heterotransplanted human tumors grown in nude mice were also successfully treated, resulting in the ultimate elimination of fibroblastic cells derived from the stroma of the tumor. The functional properties of the tumor cells (production of human chorionic gonadotropin by choriocarcinoma cells and production of carcinoembryonic antigens by pancreas carcinoma cells) were also maintained after the antiserum treatment.


Enhancement of the nuclear reactivity of alkylating agents by prednisolone. We have compared the nuclear reactions of the alkylating agents chlorambucil, melphalan, and cyclophosphamide used alone, with those produced when the same drugs were used in combination with the corticosteroid prednisolone. Studies were performed in vivo in Wistar rats bearing ascites forms of either the Yoshida sarcoma or the Walker carcinosarcoma. Strains of both tumor lines, which were sensitive to alkylating agents, showed loss of condensed chromatin, increased phosphorylation of nuclear proteins, and increased DNA cross-linking after exposure to the alkylating agents alone; these changes were generally enhanced in the presence of prednisolone. When paired tumor lines which exhibited acquired resistance to alkylating agents were studied, no nuclear changes were detected when individual alkylating agents were given alone. However, in combination with prednisolone, each of the three alkylating agents produced loss of condensed chromatin, increased nuclear protein phosphorylation, and DNA cross-linking. These chromatin changes were associated with tumor cell death and may determine drug sensitivity in the tumors studied.


Prolonged retention of estradiol by human breast cancer cells in tissue culture. Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [3H]estradiol (3 to 5 nM) and then were washed with 3 successive exchanges of medium 3, 17, and 24 or 48 hr following incubation with [3H]estradiol. The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture; (a) the concentration of [3H]estradiol and [3H]estradiol metabolites in the media washes; (b) the intracellular concentration of [3H]estradiol and [3H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidine incorporation. The [3H]estradiol concentration in the final medium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78 hr of wash. The intracellular concentration of specifically bound [3H]estradiol was initially 18 nM, and after 78 hr of wash, it was 2.8 nM. After 48 hr of wash, nearly all specifically bound [3H]estradiol was present in the nucleus. Following incubation of the cells with 5 nM estradiol and an identical wash procedure, estrogenic activity as measured by a stimulation of thymidine incorporation was observed throughout the 78 hr monitored. When 10(-6) M tamoxifen or 10(-7) M unlabeled estradiol was included in the medium washes, the washout of nonspecific binding was unaffected; however, specifically bound [3H]estradiol was essentially eliminated within 24 hr. When bovine serum albumin was included in the medium washes, total, nonspecific, and specific [3H]estradiol binding was reduced in a parallel and dose-dependent fashion. After 48 hr, cells washed with medium containing 3.5 or 7% bovine serum albumin contained one-tenth of the [3H]estradiol present in cells washed with medium alone. We conclude that medium exchanges alone do not effectively remove estradiol from MCF-7 cells, and suggest that estrogen retention by estrogen-responsive cells may mask in vitro assessments of such responsiveness in this and other systems. Inclusion of bovine serum albumin in the washes may alleviate this problem.


Arming of normal leukocytes with sera from patients with adenocarcinoma of the prostate. A tumor-associated antigen-induced leukocyte adherence inhibition assay was used to evaluate the effect of serum from patients with adenocarcinoma of the prostate on the antitumor reactivity of normal leukocytes. Peripheral blood leukocytes from 53 normal (control) subjects were armed with serum from 22 patients with localized (Stage A) and metastatic (Stage D) prostatic cancer and reacted with allogenic extract of malignant prostate as specific tumor-associated antigen. Leukocytes pre-treated with serum from patients with Stage A cancer show significantly stronger responses to malignant prostate than do those pretreated with serum from patients with Stage D cancer, which induced little or no response. This may be attributed to an "arming factor" present in the sera of patients with an initial stage of prostatic cancer which appears to be capable of sensitizing normal leukocytes and making them specifically reactive to tumor extract. The specificity of arming with individual and pooled patient's sera was delineated by the use of extracts from other genitourinary tumors.


Inhibition of phorbol myristate acetate and phytohemagglutinin stimulation of human lymphocytes by retinol. Retinol (vitamin A) in concentrations above (5 microgram/ml inhibits the stimulation of human lymphocytes by phytohemagglutinin and phorbol myristate acetate. This is in contrast to bovine lymphocytes in which retinol inhibits only phorbol myristate acetate stimulation of lymphocytes. The cause of the inhibition does not appear to be due to a direct toxic action because the viability and ultrastructure of lymphocytes exposed to a concentration of 20 microgram/ml for 72 hr were normal. The mechanism of action of the retinol is not known, but it may be mediated by a retinol-binding protein in the cells.


An estimate of the maximum in vivo covalent binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to rat liver protein, ribosomal RNA, and DNA. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an extraordinarily potent toxin, has recently been found to be a potent carcinogen producing mucosal, lung, and liver tumors in female rats. In light of this carcinogenicity, we reexamined the in vivo covalent binding of [3H]TCDD to rat liver macromolecules. Immature Sprague-Dawley rats, receiving [3H]TCDD (0.87 mCi/kg; specific activity, 39 Ci/mmol) concentrated 18 to 64% of the total administered dose in their livers, but virtually all of this radioactivity (greater than 99.9%) was extractable. The maximum unextractable radioactivity was: protein, 60 pmol TCDD per mol of amino acid residue; rRNA, 12 pmol TCDD per mol of nucleotide residue; and DNA, 6 pmol TCDD per mol of nucleotide residue. If one assumes that this small residual amount of radioactivity represents covalent binding, this binding is 4 to 6 orders of magnitude lower than that of most chemical carcinogens, and the binding to DNA is equivalent to one molecule of TCDD per DNA, equivalent to 35 cells. The results suggest it is unlikely that TCDD-induced oncogenesis is through a mechanism of covalent binding to DNA and somatic mutation.


Early effects of dimethylnitrosamine on protein chain initiation and postmicrosomal polyadenylic acid-containing RNA content in mouse liver. A mouse liver S-30 system was used to study the early effects of dimethylnitrosamine (DMNA) on polypeptide chain initiation and messenger RNA content. The inhibition of protein synthesis after DMNA administration was associated with a reduced capacity of the S-30 system to form 80S ribosomal initiation complexes. The binding of formylatable methionyl transfer RNA to polysomes was also depressed. The initiation defect was detectable in the assay system slightly later than the decrease in protein synthesis. Addition of mRNA stimulated both translation and 80S initiation complex formation but could not fully restore the activity of the S-30 system from DMNA-treated mice. A loss of poly(A)+ RNA from the postmicrosomal subfraction of the S-30 fraction was observed as early as 15 min after DMNA administration. Later, polyriboadenylic acid also decreased in the microsomal fraction. Monosomes accumulating in response to DMNA treatment were deficient in mRNA as measured by polyriboadenylic acid analysis. Conversely, the proportion of polyriboadenylic acid in the remaining polysomes increased, indicating that the mRNA had become less densely occupied with ribosomes.


Agglutination of bladder cells by concanavalin A during the early phase of treatment of rats with N-butyl-N-(4-hydroxybutyl)nitrosamine. N-Butyl-N-(4-hydroxybutyl)nitrosamine was given to male Wistar rats at a dose of 0.05% in the drinking water for one to five weeks, and agglutination of cell isolated from their bladder by concanavalin A (Con A) was determined at intervals during and after treatment. Mucosal cells were isolated from everted bladder by ethylenediaminetetraacetate treatment and sonication. As early as one week after the start of treatment, Con A caused some agglutination of isolated bladder cells, and this agglutination increased with time, reaching an almost constant value from the third week. Con A agglutination of bladder cells induced by N-butyl-N-(4-hydroxybutyl)nitrosamine treatment for only one week appeared to be irreversible, and it was still observed two weeks after the end of treatment. Scanning electron microscopy showed that microvilli developed on the luminal surface of mucosal cells in situ at the time when the isolated cells became agglutinable with Con A. Measurement of agglutinability of isolated bladder cells with Con A might be a useful way of detecting very early changes in bladder carcinogenesis.


Distribution of radiolabeled alloantibodies in mice bearing 3-methylcholanthrene-induced sarcoma. The distribution of purified 125I-labeled alloantibodies, prepared from the serum of DBA/2J mice obtained after immunization with C3H/HeJ spleen cells, was studied in immunosuppressed DBA/2J mice bearing either allogeneic C3H/HeJ 3-methylcholanthrene sarcomas growing s.c. or syngeneic SaD2 3-methylcholanthrene sarcomas. Once purified radiolabeled antibody was isolated from 125I-labeled immune gamma-globulin by a single adsorption onto C3H/HeJ RBC and elution from stroma prepared from these cells, by using 0.1 M glycine buffer (pH 3.0). Twice-purified alloantibody was then produced by Bio-Gel P-200 or Sephadex G-200 gel filtration chromatography or DEAE A-50 ion-exchange chromatography. In vitro, such purified antibodies bound specifically to C3H/HeJ RBC. In vivo, they localized to a significant extent following i.p. injection, preferentially in C3H/HeJ 3-methylcholanthrene sarcomas (4.4 to 8.9% of the injected dose per g of tumor equal to 1% of mouse weight), with mean tumor/blood ratios of 4.0 to 7.8, at 24 or 48 hr after injection. The percentage of injected dose localized in tumor and the tumor/blood ratio did not show significant differences with respect to time or method of antibody purification. Normal tissue/blood ratios in C3H/HeJ or SaD2 sarcoma-bearing mice were less than 0.9. The tumor/blood ratios in SaD2 sarcomas were approximately 0.6. Injection of 131I-labeled normal DBA/2J gamma-globulin resulted in normal tissue/blood and tumor/blood ratios of less than 0.9 in C3H/HeJ tumor-bearing mice.


Metabolic activation of arylhydroxamic acids by N-O-acyltransferase of rat mammary gland. The lactating mammary glands of rats contain an arylhydroxamic acid N,O-acyltransferase that catalyzes the formation of arylamine-substituted nucleic acid on incubation with N-hydroxy-N-2-acetylaminofluorene or N-hydroxy-N-4-acetylaminobiphenyl and transfer RNA. The acyltransferase activity migrates as a single component with a molecular weight of 28,000 on gel filtration on Sephadex G-100. Acyltransferase activities of the lactating mammary glands of Sprague-Dawley animals are approximately twice those of the less susceptible Fischer strain as determined by assay with either hydroxamic acid. The fluorene substrate was 15 times as efficient as the biphenyl compound in promoting adduct formation. Ribosomal RNA adducts formed in vivo after administration of N-hydroxy-N-2-acetylaminofluorene were consistent with an acyltransferase mechanism of activation in that the adducts did not retain the acetyl group.


beta-Hexosaminidase isoenzymes in tissues, cultured cells, and media from human fetal intestine and colonic adenocarcinoma. Isoelectric focusing of tissue homogenates revealed a predominance of beta-hexosaminidase B in colonic adenocarcinoma, whereas beta-hexosaminidase A was greater in paired normal-appearing colonic mucosa from the same patients as well as in a specimen of human fetal colonic mucosa. Because of the recognized cellular heterogeneity of these tissues, our studies were extended to an examination of these isoenzymes in 20 cultured epithelial cell lines derived from human fetal intestine and colonic carcinoma as well as the secreted enzymes in their culture media. While the B:A isoenzyme ratio was higher in human cancer cells as compared to fetal cells, some of the cancer cell lines had a greater proportion of the A isoenzyme. Examination of the isoenzyme profiles in the media of these cells revealed a greater B:A ratio whether of fetal or cancer cell origin. These studies parallel the apparent biological differences of neoplastic colonic epithelium occurring in vivo and are reminiscent of reported oncodevelopmental changes in enzymatic properties present in some malignant tissues. The differential stabilities of these two isoenzymes derived from the culture media of both types of cell lines in vitro may limit their value as markers of human colonic neoplasia.


The N-hydroxy metabolites of N-methyl-4-aminoazobenzene and related dyes as proximate carcinogens in the rat and mouse. The carcinogenicities for rats and mice of N-methyl-4-aminoazobenzene (MAB) and its hepatic microsomal metabolite N-hydroxy-N-methyl-4-aminoazobenzene (N-hydroxy-MAB) were compared under several conditions. N-Ethyl-4-aminoazobenzene, 4-aminoazobenzene, and their N-hydroxy derivatives were also included in some of the assays. About 25% of the rats given MAB or N-hydroxy-MAB (3 to 5 mmol/kg body weight) by stomach tube over a 5-week period developed hepatic tumors by 18 to 22 months. Similarly treated rats subsequently given phenobarbital in the drinking water until the termination of the experiment developed about twice as many hepatic tumors. N-Hydroxy-MAB, administered p.o., but not MAB, also induced multiple papillomas and extensive carcinomas of the forestomach in approximately 50% of the rats. Only low incidence of hepatocellular carcinomas occurred in partially hepatectomized rats given a single i.p. injection of 180 mumol/kg body weight of MAB or N-hydroxy-MAB with or without subsequent administration of phenobarbital. Although repeated s.c. doses of N-benzoyloxy-N-methyl-4-aminoazobenzene induced sarcomas at the injection site in 90% of the rats, only 3 of 20 rats developed sarcomas at the site of s.c. injections of N-hydroxy-MAB. N-Ethyl-4-aminoazobenzene, 4-aminoazobenzene, and their N-hydroxy derivatives did not induce significant numbers of tumors in any of the above assay systems. Administration to preweaning male mice of MAB, N-hydroxy-MAB, N-hydroxy-N-ethyl-4-aminoazobenzene, and N-hydroxy-4-aminoazobenzene resulted in high incidences and high multiplicities of hepatic tumors (averages of 5 to 7 tumors/mouse) within 1 year. N-Ethyl-4-aminoazobenzene and 4-aminoazobenzene also induced hepatic tumors under the same conditions, but they were less active. These data support the conclusion that the N-hydroxy metabolites of these aminoazo dyes are proximate carcinogens.


Serine hydroxymethyltransferase activity and serine incorporation in leukocytes. Studies of serine hydroxymethyltransferase activity in extracts of leukocytes from normal and leukemic subjects showed that the enzyme is present in lymphocytes and granulocytes but that activity is higher in lymphocytes. It is also higher than normal in lymphocytes from patients with chronic lymphocytic leukemia and to a lesser extent in the leukocytes of patients with acute myelocytic leukemia and acute lymphocytic leukemia. A striking increase in activity occurs in lymphocytes stimulated by phytohemagglutinin to divide in culture. Enzyme activity rises severalfold before cell number increases. Stimulated lymphocytes take up [3-14C]serine from the medium and incorporate its radioactivity into DNA, RNA, and other cell fractions. The rate of incorporation increases sharply before the rise in cell number. Thus, serine hydroxymethyltransferase activity and serine incorporation in vivo show a temporal correlation in stimulated lymphocytes. Inhibitors of DNA synthesis (e.g., fluorodeoxyuridine or high concentrations of adenosine or thymidine) block incorporation of serine radioactivity into DNA and other cell fractions. The results suggest that serine hydroxymethyltransferase activity and cellular uptake of serine have a significant role in proliferating cells.


Enhancement of antitumor activity of 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine and Baker's antifol (triazinate) with carboxypeptidase G1. Carboxypeptidase G1 (CPDG1), an enzyme that degrades folates but not the nonclassical folate antagonists triazinate (TZT, Baker's antifol) and 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP), enhanced the antineoplastic activity of these antifolates. In 6-day cell culture experiments with Walker 256 carcinosarcoma, CPDG1 at levels up to 0.54 unit/ml showed very little inhibitory effect on growth. In the presence of 10(-7) M DDMP, the grown of Walker 256 cells was similar to that of controls, but in combination with CPDG1 (0.1 unit/ml) 80% growth inhibition was observed. TZT at a concentration of 10(-8) M did not affect the growth of Walker 256 cells. The combination of 10(-8) M TZT with CPDG1 (0.1 unit/ml), however, strongly inhibited cell growth. In experiments with rats bearing Walker 256 carcinosarcoma, the administration of CPDG1 (800 units/kg/day) from Day 1 to Day 6 resulted in no significant increase in life span. Administration of TZT in doses up to 0.05 mg/kg on Day 1 or that of DDMP up to 15 mg/kg on Days 1, 3, and 5 had no antitumor effects as measured by survival of the animals. However, CPDG1 (800 units/kg/day) from Day 1 to Day 6 in combination with TZT (0.05 mg/kg on Day 1) or DDMP (15 mg/kg on Days 1, 3, and 5) resulted in increases of 50 and 30%, respectively, in the survival of the tumor-bearing animals. These results demonstrate that CPDG1 enhances the antitumor effects of TZT or DDMP.


An improved method for analyzing survival data from combination chemotherapy experiments. In this paper, we present a new method for analyzing survival data from combination chemotherapy experiments. The analysis consists of relating survival to the dosage level of each drug in the combination and using response surface techniques to determine the importance of drug interactions and to estimate optimal doses. A combination experiment using cyclophosphamide, mechlorethamine, and mitomycin C in early L1210 leukemia, advanced L1210 leukemia, and advanced P388 leukemia is used to illustrate the analyses. A therapeutic synergism has been shown. As a result of the various drug interactions, the predicted optimal dose of mitomycin C is found to be zero. This result was duplicated in each tumor system studied.


Enhancement of the thermal response of animal tumors by Corynebacterium parvum. The effect of Corynecbacterium parvum treatment on the thermal response of animal tumors was studied. Tumors were methylcholanthrene-induced (FSa-II) and spontaneous (FSa-I) fibrosarcomas in C3Hf/Sed mice. C. parvum was given i.v. and was followed by local hyperthermia at 43.5 degrees 3 days later. Cell survival determined by lung colony assays showed that preadministration of C. parvum insignificantly enhanced the thermal response of both tumors. Studies of delay of tumor growth for FSa-II demonstrated that the enhancement ratio decreased with increasing time of treatment and reached a minimum of approximately equal to 1.7. The enhancement ratio for the time at hyperthermia which achieved tumor control in one-half of the treated tumors was 1.7. Together with our previous results on normal tissue responses, the therapeutic gain factor for obtaining 50% tumor control was found to be 1.1 (1.7/1.55) for the weekly immunogenic FSa-II tumor, while it was 2.3 for moderately immunogenic FSa-I as reported previously.


Enhancing effect of thyroxine on tumor growth and metastases in syngeneic mouse tumor systems. We studied the effect of thyroxine treatment on tumor growth and metastases resulting from tumor implants on the hind feet of mice in two syngeneic systems. In control, untreated A/Jax mice, tumor Sarcoma 1 at Day 14 after implantation had average tumor weight of 582 +/- 60 (S.D.) mg and showed an incidence of 57% metastases to regional popliteal nodes and 5% metastases to thymus. In contrast, the thyroxine-treated group (40 microgram/mouse s.c., 5 times/week for 1 month) had an average tumor weight of 808 +/- 56 mg (p less than 0.001), and metastases to popliteal nodes and thymus were 90 and 35%, respectively. In another syngeneic tumor system, Lewis fibrosarcoma was implanted in C57BL/6J mice, and the tumor weight and metastatic index (derived from the number and size of the pulmonary tumor foci) were determined at Day 28. Again, the synthetic L-thyroxine treated group showed a significant enhancement tumor growth and metastatic index. The mean tumor weight in the treated group was 385 +/- 26 mg (control, 694 +/- 25 mg; p less than 0.005) and metastatic index was 84 +/- 29 (control, 30 +/- 25; p less than 0.001). Induced hypothyroidism (treatment with 131I, 100 microCi/mouse i.p.) showed the reverse effect on both tumor systems. These results suggest that both tumor systems are dependent on thyroid hormones for their growth and spread.


Mutagen-induced disturbances in the DNA of human lymphocytes detected by antinucleoside antibodies. The alkylating mutagens N-methyl-N'-nitro-N-nitrosoguanidine, methyl methanesulfonate, and N-nitroso-methylurea induced immunoreactivity to antinucleoside antibodies in human peripheral blood lymphocytes in vitro. This could also be detected in lymphocytes taken from a patient soon after i.v. administration of cyclophosphamide. The immunoreactivity response, which indicates denatured DNA or DNA single-strand breaks, was scored by immunofluorescent or immunoperoxidase techniques. Examination of blood from 10 normal subjects showed that 32 +/- 4% (S.E.) of resting peripheral blood lymphocytes were immunoreactive to antinucleoside antibodies. We have shown that these naturally occurring immunoreactive lymphocytes are largely accounted for by a subpopulation of thymus-derived lymphocytes bearing the Fc receptor for immunoglobulin M. The presence of these cells did not interfere with the use of peripheral blood lymphocytes for in vitro measurement of additional immunoreactivity caused by alkylating mutagens. The response proved to be dose dependent; up to 90% of lymphocytes could be rendered immunoreactive. Parallel studies with HeLa cells showed a similar dose-response relationship between mutagen action and immunoreactivity. With some agents, the immunoreactivity technique detected effects at lower concentrations than could be detected by HeLa cell survival studies. With N-nitrosomethylurea, measurement of DNA repair synthesis by [3H]thymidine autoradiography showed that in HeLa cells these two parameters of response to DNA damage increased in parallel. Our results provide a new basis for detecting the action of alkylating mutagens on human lymphocytes in vitro or in vivo.


Evaluation of ribonucleoside and deoxyribonucleoside triphosphate pools in cultured leukemia cells during exposure to methotrexate or methotrexate plus thymidine. Continuous exposure to inhibitory concentrations of methotrexate produces distinct rates of steady-state growth of murine leukemia L1210 and human leukemia CCRF-CEM cells in culture. Addition of thymidine to the medium produces reversal (6 to 40%) of this steady-state growth rate inhibition. This study utilized combinations of methotrexate and thymidine for an evaluation of the accompanying relationship between steady-state growth rate and changes in the ribo- and deoxyribonucleoside triphosphate pools. In L1210 cells exposed to methotrexate alone, the deoxythymidine 5'-phosphate (dTTP) pools decreased, whereas deoxyadenosine 5'-triphosphate, deoxyguanosine 5'-triphosphate, and deoxycytidine 5'-triphosphate (dCTP) remained relatively constant up to 70% inhibition of growth rate, with dCTP at a constant 112% of controls. The corresponding ribonucleoside triphosphates decreased only slightly. With the combination of methotrexate and thymidine resulting in up to 40% inhibition of growth rate, there was also a decrease in the dTTP pool while the other deoxyribonucleoside triphosphates remained relatively constant, and the corresponding ribonucleoside triphosphates again decreased only slightly. The dCTP pool was reduced to a constant 42% of control comparable to that produced by thymidine alone. With greater than 40% (with thymidine) or 70% (without thymidine) inhibition of growth rate, all pools decreased, but only dTTP was substantially reduced in proportion to the growth rate inhibition caused by methotrexate. The dTTP pool became depleted in spite of the presence of exogenous thymidine. Evaluation of CCRF-CEM cells indicated that inhibition of growth rate and nucleotide pool perturbations by methotrexate were similar to those observed in L1210 cells. However, in the presence of thymidine, inhibition of growth rate appeared related to decreased pools of dCTP, deoxyadenosine 5'-triphosphate, and deoxyguanosine 5'-triphosphate, rather than dTTP as was observed for L1210 cells. Hence, mammalian cells were capable of responding in a differential fashion to pharmacological perturbations, and this capacity may play a role in determining therapeutic selectivity. Since the ribonucleoside triphosphate decreases were slight and relatively uniform during methotrexate-induced perturbations, the deoxyribonucleoside triphosphate pools appear to be more directly related to inhibition of growth rate. The results are consistent with the concept that slight imbalances in the deoxyribonucleoside triphosphate pools dramatically inhibit DNA synthesis, as mediated through their interaction with DNA polymerase.


Metabolic conversion of indicine N-oxide to indicine in rabbits and humans. Indicine N-oxide, a pyrrolizidine alkaloid N-oxide that exhibits antitumor activity without some of the toxic effects associated with other pyrrolizidine alkaloids, is metabolized to indicine in rabbits and humans. Indicine can be detected in the plasma and is excreted in the urine in a dose-dependent manner following the i.v. administration of indicine N-oxide. The p.o. administration of indicine N-oxide leads to an increased plasma concentration and an increased urinary excretion of indicine. The hepatic microsomal fraction and the gut flora both catalyze the anaerobic reduction of indicine N-oxide to indicine in vitro. Whole-animal studies suggest that the gut flora play a major role in the metabolic reduction of indicine N-oxide by the rabbit. Indicine N-oxide is not actively excreted in the bile, and it is probable that indicine N-oxide finds its way into the gut by passive diffusion following i.v. administration. Neomycin and erythromycin, which reduce the number of anaerobic bacteria in the gut, lead to decreased plasma levels and a decreased urinary excretion of indicine.


Cytotoxic effects of chemotherapeutic drugs on mouse testis cells. Studies of testicular cell killing in mice by several chemotherapeutic drugs have been performed to EVALUATE THE HARMFUL EFFECTS OF ONCOLYTIC AGENTS ON REPRODUCTION. Seven drugs, Adriamycin, 1-beta-D-arabinofuranosylcytosine, bleomycin, cyclophosphamide, hydroxyurea, vinblastine, and vincristine, given as single injections, were cytotoxic to differentiated spermatogonia. Adriamycin was also highly effective in killing stem cells. The other drugs produced little or no stem cell loss even at doses toxic to the animals. Negligible killing of spermatocytes and spermatids was noted at any dose level. The results demonstrated that oncolytic agents preferntially killed cells at specific stages of the spermatogenic pathway in mice at doses within the clinical range for humans.


Induction of sarcomas in nude mice by implantation of Syrian hamster fetal cells exposed in vitro to nickel subsulfide. In vitro exposure of Syrian hamster fetal cells to nickel subsulfide (alpha Ni3S2) yielded positive colony assays for morphological transformation. A dose-response relationship was found between the concentration of alpha Ni3S2 and the incidence of morphological transformation. Exposures of alpha Ni3S2 induced morphological transformation at concentrations (0.1 or 1.0 microgram/ml of culture medium) which did not impair cell plating efficiency. Nickel monosulfide (NiS) did not induce morphological transformation of Syrian hamster fetal cells under the same conditions. Clones of alpha Ni3S2-transformed cells were able to grow in soft agar medium and demonstrated increased basal and induced activities of ornithine decarboxylase. Undifferentiated sarcomas developed in 26 of 27 nude mice at the site of s.c. injection of clones of alpha Ni3S2-transformed cells. No tumors developed in 19 control nude mice which were given s.c. injections of nontransformed Syrian hamster fetal cells which had not been exposed to alpha Ni3S2. This study demonstrates that fetal cells which undergo transformation following exposure to alpha Ni3S2 are capable of producing malignant tumors in nude mice.


Isolation and characterization of alpha-lactalbumin from a rat mammary tumor. alpha-Lactalbumin was purified to homogeneity from the R3230AC rat mammary adenocarcinoma. alpha-Lactalbumin from the R3230AC tumor had the same amino acid and carbohydrate composition, migration on gel electrophoresis, and specific activity as did alpha-lactalbumin isolated from normal rat milk.


Protection of mice against lethal dosages of nebularine by nitrobenzylthioinosine, an inhibitor of nucleoside transport. In the presence of nitrobenzylthioinosine (NBMPR) a potent inhibitor of nucleoside transport, Roswell Park Memorial Institute 6410 cells proliferating in culture were protected from otherwise inhibitory concentrations of 9-beta-D-ribofuranosylpurine (nebularine); cellular uptake of nebularine was greatly reduced under these circumstances. Initial rates of nebularine uptake by Roswell Park Memorial Institute 6410 cells were inhibited by NBMPR, indicating that the latter interfered with nebularine transport. NBMPR protected mice against potentially lethal treatment regimens with nebularine, 4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin) or 4-amino-5-cyano-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (toyocamycin); protection resulted when NBMPR was administered i.p. in advance of or simultaneously with nebularine, but not when NBMPR followed nebularine by 1 hr. Both NBMPR and its 5'-monophosphate protected mice against nebularine lethality when administered s.c.


Carcinogenesis in rat esophagus by intraperitoneal injection of different doses of methyl-n-amylnitrosamine. The carcinogenicity of methyl-n-amylnitrosamine in MRC-Wistar rats was determined after i.p. injection at a variety of dose schedules. After 6 weekly methyl-n-amylnitrosamine injections of 25 mg/kg or 12 weekly injections of either 12.5 or 25 mg/kg, the incidence of esophageal squamous cell papillomas was 85 to 100% and that of esophageal squamous cell carcinomas was 40 to 65%. With 12 injections, the mean survival time was 25 to 31 weeks. Treatment with 1 or 2 doses of 50 mg/kg produced a lesser incidence (less than 20%) of esophageal tumors, with a longer survival time of 67 to 77 weeks. One 85-mg/kg injection caused esophageal carcinomas in 5 of 7 rats. The treated groups also had squamous cell papillomas and carcinomas in the nasal cavity (up to 50% incidence) and trachea (up to 30% incidence). Hence, a 6- or 12-week treatment schedule was adequate for inducing esophageal tumors and could be used for studies on agents modifying esophageal tumor induction by methyl-n-amylnitrosamine.


Quantitative models for growth inhibition of human leukemia cells by antitumor anthracycline derivatives. A batch elution method with hydroxylapatite was developed to assay DNA damage by a set of antitumor anthracycline derivatives and was standardized with respect to the kinetics of unwinding, size of the alkaline unwinding unit, and fidelity of selective elution of single and double-stranded DNA. The method was applied to a study of a set of 10 antitumor anthracycline derivatives which inhibit growth of CCRF-CEM human leukemia cells over a range of potencies exceeding four orders of magnitude. The derivatives, including Adriamycin, daunorubicin, and carminomycin, vary in structure at C-4 and C-13, with substitutions at C-14 and N and stereochemical differences at C-4'. In a static model (fixed drug concentrations and incubation times), the potency [1/ID37 (concentration of agent that inhibits cell growth by 37%)] of nine of the ten derivatives may be expressed as functions of DNA damage (n), inhibition of thymidine incorporation (l), and drug retention (r): ID37 = Ka(r/l . n)kb, with a coefficient of correlation of greater than 0.99. A kinetic model with 4-demethoxydaunorubicin (varying concentrations and incubation times) was also described. Following initial uptake and a period of rapid loss after cells are washed free of excess drug, the change in agent concentration in the cells follows first-order kinetics. The cell index (cell number after 50 hr in drug-free growth medium/cell number after initial 2-hr exposure with drug) may be expressed linearly in terms of the kinetics of drug loss (coefficient of correlation, greater than 0.98), or as functions of 1/n (coefficient of correlation, greater than 0.958), 1/l . n (coefficient of correlation, greater than 0.963, or r/l . n (coefficient of correlation, greater than 0.963). These studies may be used to define a class of similarly acting anthracycline agents and to give some insight into the mechanism of action of the agents that fall within the class.


Ultrastructure of doxorubicin (adriamycin)-induced skin ulcers in rats. Skin necrosis was produced in 24 male Fischer 344 rats by intradermal injection of 0.5 ml of doxorubicin (Adriamycin) at a concentration of 2 mg/ml. The resulting wounds healed slowly over 6 to 7 weeks with the reduced contraction rate paralleling the prolonged morbidity of doxorubicin ulcers in humans. Electron microscopy showed bizarre rough endoplasmic reticulum, double-walled  vacuoles, and swollen mitochondria from 1 through 12 weeks after injury. Myofibroblasts with 60- to 80-A microfilaments with electron-dense bodies, intercellular connections, and prominent microtubules were seen from 4 through 12 weeks after injury. Although the appearance of myofibroblasts was delayed, their structure was normal. The delayed contraction of doxorubicin-induced skin ulcers thus appears due to persistent nonspecific cellular damage at the nuclear level rather than to specific derangement of myofibroblast function.


Photodynamic inactivation of synchronized human cells in vitro in the presence of hematoporphyrin. Dose-response curves for inactivation of synchronized cells from the human cell line NHK 3025 in the presence of hematoporphyrin and light are shown. Sensitivity toward photodynamic inactivation is increasing as the cells move in the cell cycle from G1 to mid-S. The increase is mostly due to a change in the extent of the shoulder of the dose-response curve at low doses. The quasithreshold dose (Dq) is high in early G1 and much lower in mid-S. No great variations were found in the slope (1/Do) of the dose-response curves at high doses. The shape of the dose-response curves indicates a higher capacity for repair of sublethal damage in the early part of the cell cycle than in the later stages.


Effects of daunorubicin and doxorubicin, free and associated with DNA, on hemopoietic stem cells. We have compared daunorubicin (DNR)-DNA with free DNR and doxorubicin (DOX)-DNA with free DOX for their effects in vivo in mice on pluripotent stem cells and granulocytic committed stem cells. Dose-survival, time-survival, and recovery curves were obtained after one i.v. injection of either drug. The dose-survival curves of colony-forming units-spleen (CFU-S) and colony-forming units-committed stem cells (CFU-C) were exponential in shape with both agents. DNR-DNA appeared more toxic to the hemopoietic precursor cells than did free DNR. In contrast, DOX-DNA was less toxic toward CFU-S and as toxic as DOX toward CFU-C. Time-survival curves indicated a minimum level of CFU-S and CFU-C at about 33 hr. After that, the recovery of CFU-S was rapid for DNR-treated mice but remained below 50% of the controls on Day 12 for the DNR-DNA-treated group. In mice previously given injections of DOX or DOX-DNA, the recovery of the CFU-S was more protracted in time with a better recovery in mice treated with DOX-DNA. Both DNR and DNR-DNA induced an initial CFU-C decrease followed by a rapid but transient rise with a maximum on Day 4 after chemotherapy. On Day 12, the CFU-C recovery was still incomplete in both DNR- and DNR-DNA-treated mice. In the groups treated with DOX, the CFU-C recovery was more important after DOX-DNA complex than after free DOX. The results are discussed in view of the "lysosomotropic chemotherapy" hypothesis.


Quantitation of intracellular metabolites of [35S]-6-mercaptopurine in L5178Y cells grown in time-course incubates. 6-Mercaptopurine (6MP) metabolism was quantitatively determined in L5178Y murine lymphoma. Cells grown in time-course incubates with [35S]-6MP were extracted with cold perchloric acid, and the buffered extracts were subjected to high-performance liquid cation-exchange chromatography prior to and after hydrolysis with alkaline phosphatase. Free sulfate, 6-thiouric acid, 6-thioxanthosine, 6-thioguanosine, 6-thioinosine, free 6MP, and 6-methylthioinosine were separated from each other; identified in the radiochromatograms by elution volume, UV spectroscopic data, and enzymatic peak-shifting analyses with purine nucleoside phosphorylase; and quantitatively determined by means of 35S radioactivity. Gross intracellular 35S concentrations remained constant at 5 x 10(-5) M after 1 hr of incubation. 6MP metabolism in L5178Y cells was distinguished into an early phase (to 1 hr of incubation) in which 6MP was predominantly catabolized to 6-thiouric acid and free sulfate, into an intermediate phase (to 8 hr) in which substantial amounts of free 6MP and of ribonucleotides of 6-thioxanthosine and 6-thioguanosine were present while the concentrations of nonnucleotide oxidation products sharply decreased, and into a late phase (to 24 hr) in which the ribonucleotides of 6MP, of 6-thioguanosine and, in particular, of 6-methylthioinosine were the most abundant metabolites.


Effect of dietary fiber on the induction of colorectal tumors and fecal beta-glucuronidase activity in the rat. The purpose of the present study was to investigate whether three different types of dietary fiber, wheat bran, carrot fiber, and citrus pectin, influenced the induction of colorectal tumors produced by 1,2-dimethylhydrazine in rats. In all groups, the tumor yield was high (87 to 97%). In the wheat bran and carrot fiber groups, the incidence of colorectal tumors was not significantly different from that of the group fed on the fiber-free basic diet. The citrus pectin group, however, had a significantly higher incidence of colorectal tumors (p less than 0.001). An increased number of auditory duct tumors was also noted in this group. In a separate experiment, dietary pectin induced a 10-fold increase in fecal beta-glucuronidase activity but did not alter this activity in the bowel wall. It has been suggested that dietary fiber protects against the induction of colorectal tumors, but this was not the case in the experiment. It is possible that the high tumor yield made the demonstration of a weak protective effect of wheat bran impossible. The reason for the increased occurrence of tumors in the citrus pectin group is obscure and will be subjected to further investigation. Fecal beta-glucuronidase activity might be one factor of importance in the activation of the carcinogen.


Luminal plasma membrane organization in rat urinary bladder urothelium after direct exposure in vivo to N-methyl-N-nitrosourea. Adult female rat urinary bladders were exposed to N-methyl-N-nitrosourea in vivo. After 12 weeks, urothelial hyperplasia was established. Ultrastructural changes of the luminal plasma membrane were studied with thin sectioning and freeze-fracturing techniques. The mosaic pattern which is a characteristic specialization of the normal luminal plasma membrane and of cytoplasmic vesicles disappeared. The plasma membrane of the transformed urothelium was indistinguishable from an undifferentiated membrane in the following respects: thickness; apparent symmetry in cross-section; and distribution of intramembranous particles. Zonulae occludentes became permeable to colloidal lanthanum and appeared disassembled, while desmosomes developed concomitantly. These membrane changes implicate increased permeability and less adaptability of urothelial function to mechanical stress caused by bladder volume changes. The increased desmosome formation may indicate that squamous metaplasia is a feature of early malignant urothelium transformation. It is proposed that N-methyl-N-nitrosourea treatment induces a change in the plasma membrane assembly and specialization.