Showing papers in "Cancer Research in 1973"

Morphology and Growth, Tumorigenicity, and Cytogenetics of Human Neuroblastoma Cells in Continuous Culture

Abstract: Summary Continuous cell lines, SK-N-SH and SK-N-MC, were established in cell culture from human metastatic neuroblastoma tissue and maintained in vitro for 1 to 2 years. SK-N-SH comprises two morphologically distinctive cell types, a small spiny cell and a large epithelioid cell. SK-N-MC is composed of small fibroblast-like cells with scant cytoplasm. In monolayer culture both cell lines form disoriented growth patterns and reach high saturation densities. Population-doubling times were 44 and 32 hr for SK-N-SH and SK-N-MC, respectively. Inoculum levels of 10 7 cells of both lines produced tumors confirmed by histopathological examination, at frequencies of 30 to 40% in cheek pouches of conditioned Syrian hamsters. SK-N-SH cells are characterized by high dopamine-β-hydroxylase activity while SK-N-MC cells have no detectable activity. However, for SK-N-MC but not SK-N-SH, the presence of intracellular catecholamine was indicated by formaldehyde-induced fluorescence. The lines are near-diploid with several chromosomal markers; SK-N-MC cells contain double- minute chromosomes. Growth, biochemical, and cytogenetic properties confirmed that the lines comprise malignant cells of neurogenic origin.

Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division.

Abstract: A line of C3H mouse embryo cells highly sensitive to postconfluence inhibition of cell division, designated C3H/10T1/2, has been established, and a clone from this line (clone 8) has been characterized at early and late passages (200 to 450 days in culture). The cells of clone 8 are approximately 1730 cu µm in volume, their plating efficiency is 12 to 30%, their average generation time is 15.5 hr, and their saturation density is 2.9 to 3.8 × 104 cells/sq cm. The cell survival is 30% after freezing in 10% dimethyl sulfoxide and storage in liquid nitrogen. The cells of this clone are negative with respect to the spontaneous expression of C-type RNA murine viruses and viral antigens. Tests for mycoplasma contamination are negative. All the cells of this line are aneuploid with a stable mode of 81 chromosomes (40 to 60% of cells). Tests for tumorigenicity at all passages were negative. No spontaneous transformation in vitro has been observed in the stock cultures transferred on a regular schedule.

Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division.

Abstract: A line of C3H mouse embryo cells (C3H/10T1/2 clone 8) sensitive to postconfluence inhibition of cell division was used for a variety of quantitative and qualitative studies of chemical oncogenesis in culture. The polycyclic hydrocarbons (PH), 3-methylcholanthrene, dibenz[a,h]anthracene, and 7,12-dimethylbenz[a]anthracene, caused varying degrees of cytotoxicity and produced morphologically and malignantly transformed foci in these cells with a dose-dependent frequency, while N -methyl- N ′-nitro- N -nitrosoguanidine was toxic but did not transform under the same conditions. Transformation frequency was related to cell density at the time of treatment and to the duration of treatment with PH. After treatment of the cells with the PH, three types of morphologically altered foci were identified. Foci of each type were cloned and inoculated into irradiated syngeneic mice. Two of the three types of foci gave rise to fibrosarcomas when cells were inoculated at Passages 2 to 4 after cloning. The determination of transformation frequency in this system includes only these two types of malignantly transformed foci. The third type of focus was morphologically minimally altered but did not give rise to tumors under the same conditions. Control cells did not produce tumors. The saturation densities of several transformed lines were shown to be greater than the control line and did not correlate with the growth rate of the cells. The antimycotic agent amphotericin B (Fungizone) interfered with transformation when it was present in the medium at the time of exposure of the cells to the PH.

A high susceptibility of Fanconi's anemia to chromosome breakage by DNA cross-linking agents.

Abstract: Summary Peripheral blood lymphocytes of patients with Fanconi9s anemia (FA) were tested for their susceptibility to chromosome breakage by caffeine, chloramphenicol, actinomycin D, methylmethanesulfonate, nitrogen mustard, mitomycin C, decarbamoyl mitomycin C, N -methyl- N′ -nitro- N -nitrosoguanidine, 4-nitroquinoline 1-oxide, 8-methoxypsoralen, and 60 Co γ-ray and ultiviolet irradiations. A definitely abnormal response of the chromosomes of the FA cells was found when they were treated with nitrogen mustard and mitomycin C and after the irradiation with long-wavelength ultraviolet light in the presence of 8-methoxypsoralen. The specifically increased susceptibility to these compounds, which can introduce interstrand cross-links into DNA, is interpreted as an indication that the FA cells are defective in the repair mechanism to tolerate the cross-links produced in their DNA. The impairment of the capacity to tolerate the lesions produced in DNA may be implicated in the increased risk to develop malignant neoplasms in this hereditary disorder.

Clinical and pharmacological studies with cis-diamminedichloroplatinum (II).

Abstract: cis -Diamminedichloroplatinum(II) is the first of a group of platinum coordination complexes with antineoplastic activity to be studied in humans. This Phase I investigation characterizes the toxicity and pharmacological disposition of the drug in 10 patients. Plasma levels of cis -diamminedichloroplatinum(II) decayed in a biphasic mode, with an initial half-life of 25 to 49 min and a secondary phase ranging from 58 to 73 hr. Protein binding exceeded 90% of radioactivity in this phase. Intracellular leukocyte levels approximated 6 to 11% of coincident plasma samples. Urinary excretion was incomplete, with only 27 to 45% of radioactivity excreted in the first 5 days. The initial fractions of radioactivity were largely unchanged drug, although this changed with time. The incorporation of thymidine-3H into DNA was inhibited in leukemic leukocytes only after prolonged exposure in vitro to cis -diamminedichloroplatinum(II). Acute lymphocytic cells appeared more sensitive than myelocytic cells in vitro , although the only objective antineoplastic response noted was a transient decrease in blast count in a patient with acute myelocytic leukemia. Renal impairment was the dose-limiting toxicity in the single-dose escalation scheme used. Rises in serum creatinine occurred in three of six patients who received doses of 1.95 mg or more per kg, and progressive renal failure contributed to the death of one patient.

Reactivity of Lymphocytes from Normal Persons on Cultured Tumor Cells

Abstract: Summary Cultured tumor cells from 7 established lines and 12 short-term cultures were reacted with lymphocytes from patients with the same “histological type” of cancer as the target cells in 995 tests and with lymphocytes from normal controls in 1099 tests. The average reactivity was significantly greater with lymphocytes from normal persons in 3 of the 7 established lines and 2 of 12 short-term cultures. In only 1 short-term bladder tumor culture was there indication of greater cytotoxicity produced by lymphocytes from bladder cancer patients than by cells from normal persons. Cell-mediated target cell reduction of cultured human tumor cells is not confined to patients with cancer.

A comparison review of key epidemiological studies in cervical cancer related to current searches for transmissible agents.

Abstract: Summary The search for a venereally transmissible oncogenic agent which may show relatedness to onset of cervical cancer rests upon three main evidential currents: demographic demonstration of cultural variables associated with increased risk, epidemiological focus upon sexual factors maximizing risk, and the assumption that cervical cancer begins and develops according to a multistage continuum. For this, an abbreviated model conforming multistage carcinogenesis in the human uterine cervix with the classical animal model introduces an epidemiological review. Assumptions required for this rationale include maximal biological activity in the cervical cellular matrix during adolescence, an oncogenic agent for initiation of neoplasia, and recognition of pivotal events directly related to risk. In reviewing variables and attributes described in the literature, distinction is made between those that are cultural and indirect and those that are biological and may directly transform epithelium. A set of comparison studies is presented in assessing historically described major influences upon risk and each is examined for relevance, using a comparison ratio for detection of excessive frequencies between patients and controls. It is shown that onset of sexuality before age 17 is the most powerfully discriminating variable in virtually all studies where this has been investigated and that a history of multiple sexual consorts is a supporting variable of some strength. Marital variables and those associated with sociosexual stability are cultural and relate to the biological variables of early coitus and multiple sexual partners. Coital frequencies, menstrual patterns, and gravidity seem to bear no discernible risk relationship. Noncircumcision of sexual partners is shown to have been limited in most studies to husbands and in some studies to selected or all coital mates, yet no study reveals differences between patients and controls for exposure to uncircumcised partners. Comparison studies are collected in charts to demonstrate relative strength and consistency of trends. Cervical cancer is the only solid neoplasm for which epidemiological findings in humans have been adapted to a model developed with laboratory animals, and it is the only tumor that has been studied with regard to a full universe of possible variables and attributes that might alter risk. This has resulted in the currently espoused hypothesis that Herpesvirus hominis type 2 may be an initiating or promoting carcinogenic agent which can be transmitted during coitus by the malc donor to the female host at risk.

Clinical pharmacology of cyclophosphamide.

Abstract: Summary The human pharmacology of cyclophosphamide was investigated in 26 patients who received cyclophosphamide 14 C in doses of 6 to 80 mg/kg i.v. Levels of the intact drug in plasma and urine and excretion of 14 C label in breath and stools were determined by liquid scintillation counting. Plasma and urine alkylating activity was measured by reaction with 4-(4-nitrobenzyl)pyridine. Protein binding of cyclophosphamide and plasma alkylating metabolites were determined by plasma ultrafiltration. Injected cyclophosphamide distributed rapidly into 64% of body weight, and plasma cyclophosphamide half-life in patients without prior drug exposure was 6.5 hr. Not more than 20% of injected cyclophosphamide was excreted intact in urine at any dose level. Plasma alkylating metabolites were 56% bound to plasma proteins. After a 40-mg/kg dose, peak unbound alkylating activity averaged 13.3 mµmoles/ml, and in most patients at this dose alkylating activity in the plasma was measurable for 24 hr. Sixty-eight % of injected 14 C label was excreted in urine. Breath and fecal excretion were negligible. In a regimen of five consecutive daily cyclophosphamide administrations, cyclophosphamide half-life was shorter and peak alkylating levels were constantly higher on the 5th day than on the 1st day. Prior patient treatment with allopurinol resulted in significantly longer cyclophosphamide half-life, but concomitant prednisolone treatment had no effect. The effect of hepatic metastases on cyclophosphamide metabolism was unclear. Moderate renal failure in one patient resulted in prolonged retention of alkylating materials in plasma and severe toxicity. Although patients with and without prior exposure to microsomal enzyme-inducing drugs demonstrated marked variation in plasma cyclophosphamide half-life and peak alkylating levels, the total concentration × time product remained relatively constant for a given cyclophosphamide dose, suggesting that alterations in the rate of cyclophosphamide metabolism by drugs or liver metastases in the absence of renal failure will not change toxicity or therapeutic effect.

Development of an Animal Brain Tumor Model and Its Response to Therapy with 1,3-Bis(2-chloroethyl)-1-nitrosourea

Abstract: Summary A chemically induced rat glioma, carried in cell culture, was transplanted to the brain of CDF rats. The tumor thrived in cell culture, producing a typical malignant astrocytoma when implanted in rat brain. Survival of tumor-bearing rats varied in different experimental groups, but the range of survival within each experimental group was within acceptable limits. The rat glioma in cell culture grew progressively through three phases, namely, exponential, stationary, and a variable phase, limited by space and medium. The cell-cycle time of the glioma in vivo was 20 hr, the growth fraction was 0.35 to 0.46, the observed doubling time was 72 hr, and the cell-loss factor was 0.42. These data, along with the chronology of the cell-cycle phases, enhance the usefulness of this system as a model for brain tumor chemotherapy. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) significantly increased the survival time of rats bearing intracerebral gliomas. A dose of BCNU approximating 40% of the 10% lethal dose, when given 9 and 16 days after tumor implantation, produced an increased life-span of 38%. A dose of BCNU equivalent to 80% of the 10% lethal dose given on Days 9 and 16 produced an increased life-span of 84%. When BCNU was withheld until the 16th and 23rd days, a significant increase in survival time was still evident. The advantages of this animal model for brain tumor chemotherapy are that (a) the transplanted tumor is of glial origin, (b) the tumor take of the model has been 100%, (c) the mean survival of animals is approximately 30 days, permitting the start of a treatment schedule after the tumor has grown to significant size, and (d) the rat is suitable for various routes of therapy.

Increased Sialic Acid Density in Surface Glycoprotein of Transformed and Malignant Cells—a General Phenomenon?

Abstract: Summary Established lines of malignant (MBVIA) and nonmalignant (MBIII) lymphoblasts, baby hamster kidney cells (BHK21/C13), BHK21/C13 cells transformed by polyoma virus, 3T3 mouse fibroblasts, 3T3 cells transformed by SV40 virus, spontaneously transformed 3T3 cells (3T3-f), rat liver cells (RLC), and Novikoff hepatoma N1S1-67 cells were studied in this investigation. Each homologous pair of proliferating cells was differentially labeled with l-fucose-14C and -3H for detection of increased sialic acid density in glycoprotein components of the transformed cell surface. The change was detectable by the faster elution from Sephadex G-50 or Bio-Gel P-10 of the trypsin- and Pronase-digested material of malignant as compared with nonmalignant cells, and the coincidence of elution profiles following neuraminidase treatment. This change was encountered in all cells studied; the greatest change was in 3T3-f cells and the smallest was in SV40-3T3 cells, in which it could be established only by gel filtration over Bio-Gel P-10. Increased sialylation of glycoprotein components has thus been demonstrated for 3 types of cells, namely, lymphoblast, fibroblast, and epithelioid cells cultured in vitro, and for 3 carcinogenic determinants, namely, viral, chemical, and “spontaneous.” We suggest that the transformed cell surface—being unable to modulate its expression as a function of the cell cycle in the manner of normal cells—is blocked in a condition of increased sialyl extension resembling that of the normal mitotic cell surface, and thus counteracts functional contract relations between cells.

Induction of adenocarcinomas of the colon in mice by weekly injections of 1,2-dimethylhydrazine.

Abstract: Summary Thirty-four CF1 mice were given weekly s.c. injections of 20 mg of 1,2-dimethylhydrazine hydrochloride per kg; this treatment induced colonic carcinomas in more than 90% of the animals after 186 days. The earliest change was found to be a widening of the proliferative compartment within the colonic crypts; at a later stage, there was an increase of the tritiated thymidine-labeling index from 9.3% at Day 45 to 16.2% at Day 87. The first histological changes were focal hyperplastic changes and focal atypias on the tip of the folds of the distal colonic mucosa (Day 38) confined to single crypts. The colonic tumors were located in the distal colon and rectum, with a high incidence of squamous cell cancers originating from the anal canal. Saline extracts of colonic tumor material precipitated specific antimouse embryonic rabbit serum. Assays of hypoxanthine phosphoribosyltransferase in the villus fraction of the small bowel of the 1,2-dimethylhydrazine hydrochloride-treated tumor-carrier mice indicated lower activity, compared to that of controls.

Vincristine treatment of advanced cancer: a cooperative study of 392 cases.

Abstract: SUMMARY Vincristine was studied in a series of dose levels in 392 patients with advanced cancer. It produced tumor regres sions in a substantial proportion of patients with advanced lymphosarcoma, reticulum cell sarcoma, Hodgkin's dis ease, breast cancer, bladder cancer, and carcinomas of un known primary site. Response occurred about as frequently at 25 jug/kg/week as at higher doses. Maximum response rates occurred in those patients who sustained moderate toxicity as contrasted to those with only mild or no toxic effects or to those with severe manifestations of drug effect on normal tissues. Dose-related sensory, motor, and auto nomie neuropathy and central nervous system dysfunc tions were observed. Leukopenia and thrombocytopenia were observed. The frequency of responses achieved and their duration were modified by good- or poor-risk status of the patients. Prediction of survival was rather accurate as determined by comparison of prognostication and actual survival times. Responders survived longer than nonresponders. In pa tients with breast cancer, the survival extension beyond prediction cannot be explained as due solely to vincristineinduced remission time. In some other diseases, therapeu tic response to vincristine was able to overbalance a poor prognosis and apparently to increase survival beyond that predicted.

Microsomal metabolism of dimethylnitrosamine and the cytochrome P-450 dependency of its activation to a mutagen.

Abstract: Summary Oxidative demethylation of the secondary carcinogen dimethylnitrosamine (DMN) by isolated mouse liver microsomes and the activation of DMN to a bacterial mutagen followed similar kinetics. The rates of demethylation and DMN activation increased following induction of the cytochrome P-450 mixed-function oxidase system by polychlorinated biphenyls. Both the oxidative demethylation and the activation of DMN to a mutagen were inhibited by carbon monoxide, and the inhibition was maximally reversed by monochromatic light at 450 nm. These observations indicate that both microsomal metabolism and activation of DMN to a mutagen are cytochrome P-450 dependent.

Alkaline elution analysis, a new approach to the study of dna single- -strand interruptions in cells.

Abstract: Summary A procedure is described whereby shortened DNA single strands can be selectively eluted from filters upon which cells have been lysed. This provides a sensitive measure of DNA single-strand breakage that may be applicable to the detection of cell damage resulting from certain types of chemotherapeutic agents. The procedure was applied to the following processes in L1210 cells: ( a ) production of single-strand breaks in DNA by X-irradiation of cells; ( b ) repair of these breaks by subsequent incubation of the cells; ( c ) transient occurrence of newly replicated DNA as relatively short chains. X-irradiation converts cell DNA from a slowly eluting to a rapidly eluting form. The conversion is first order with respect to X-ray dose and has a D 37 dose of not more than 400 rads. The DNA single-strand units affected by this first-order process have a minimum size of 1.5 × 10 9 daltons (1500 µm). Repair after an X-ray dose of 1000 rads was essentially complete within 30 min. Newly replicated DNA is in a rapidly eluting form which is slowly converted to the slowly eluting form.

Effects of Varying the Exposure to Phenobarbital on Its Enhancement of 2-Acetylaminofluorene-induced Hepatic Tumorigenesis in the Rat

Abstract: In a previous study, phenobarbital feeding enhanced hepatic tumorigenesis in rats previously fed 2-acetylamino-fluorene (AAF). The present study analyzed this enhancement by comparing tumor incidences in rats fed phenobarbital for various periods following a fixed exposure to AAF. A 5- or 20-day treatment with phenobarbital immediately after cessation of AAF feeding produced little tumorigenic enhancement, in comparison with the severalfold enhancement seen in rats receiving phenobarbital for 100 days and longer. On the other hand, the interposition of a 10- or 30-day interval between the cessation of AAF treatment and the beginning of phenobarbital treatment (which was then continued throughout the experiment) produced exhancement comparable to that produced by beginning the phenobarbital treatment immediately after cessation of AAF feeding. These results indicated that ( a ) prolonged exposure to phenobarbital was required for tumorigenic enhancement and ( b ) the tumorigenic lesion produced by AAF was relatively stable, and its expression could be enhanced by phenobarbital long after the cessation of AAF treatment. Observation of the kinetics of tumor incidence throughout the experiment showed that phenobarbital: ( a ) decreased the latent period between the end of the carcinogen treatment and the appearance of tumors; ( b ) increased the growth rate of the tumors; and ( c ) increased the rate of appearance of new tumor foci. No metastases were seen in rats given AAF alone or followed by phenobarbital, and the morphological characteristics of the tumors were similar with both types of treatment. Phenobarbital, therefore, did not appear to alter the degree of differentiation of the tumors.

Ultrastructural, Histological, and Biochemical Alterations Produced by 12-O-Tetradecanoyl-phorbol-13-acetate on Mouse Epidermis and Their Relevance to Skin Tumor Promotion

Abstract: A single application of 12- O -tetradecanoyl-phorbol-13-acetate to mouse skin induces an ordered sequence of ultrastructural changes in the cells of all layers of the epidermis, which correlate well with the changes in the rate of precursor incorporation into protein, RNA, and DNA in the whole skin and also with changes in the thickness, number of nucleated cell layers, and mitotic index of the interfollicular epidermis. The incorporation of orotic acid-5-3H into RNA is increased within 1 hr and reaches a peak by 3 hr. An increase in leucine-14C incorporation into protein is observed at 2 hr, but it is marked only after 6 to 7 hr and reaches a high plateau by 12 hr. The incorporation of thymidine-3H into DNA is markedly depressed for up to 9 hr and then sharply increases, to reach a first peak by 18 hr. The mitotic index is depressed at 8 hr, reverting to control levels at 12 to 15 hr followed by an increase in mitotic activity, and reaching a peak by 24 hr. At 16 hr, there is a marked increase in the thickness of the interfollicular epidermis and in the number of its nucleated cell layers. At this time, the mitotic index and DNA synthesis are depressed or are at control level, indicating that the increase in nucleated cell layers is due to hypertrophy, not to proliferation. Two hr after administration of the 12- O -tetradecanoyl-phorbol-13-acetate, the nucleoli of the basal cells are markedly enlarged, with an increase of the granular and fibrillar components. By 4 hr, the granular component is further increased, polysomes appear, and the intercellular spaces between basal cells are dilated. By 6 hr, the cytoplasm of the basal cells is enlarged and filled with polysomes. The mitochondria are enlarged and have more cristae and a less dense matrix. By 12 hr, the number of polysomes is further increased, and profiles of the granular reticulum and Golgi complexes are more numerous so that the stimulated basal cells resemble those of embryonic epidermis. By 24 hr, markedly hypertrophic cells and granular mitochondrial inclusions are frequently seen. By 48 hr, flattening, involution of the organelles, and accumulation of numerous keratohyalin granules occur in the cells of the upper layers. Granular electron-dense material appears in the dilated intercellular spaces at 6 hr and increases until 36 hr, when it is also in the cisternae of the endoplasmic reticulum. The superficial cells, normally flattened, are swollen by 2 hr, and they progressively regain their nuclear and cytoplasmic organization so that, by 12 hr, they resemble the stimulated basal cells. Dark cells with ultrastructural features distinct from the other epidermal cells are observed after administration of 12- O -tetradecanoyl-phorbol-13-acetate, but are not found in normal or acetone-treated controls. The relevance of the cellular events to tumor promotion is analyzed, and we propose that tumor promoters act by altering the normal differentiation pathway, thus enabling expression of the neoplastic phenotype.

Studies on the quantitative biology of hyperthermic killing of HeLa cells.

Abstract: Summary The quantitative biology of hyperthermic killing of HeLa cells was studied. Plots of cell survival versus doses of hyperthermia did not show first-order kinetics. The rate of HeLa cell killing shows a striking temperature-dependent relationship in the 41.0–43.0° temperature range. There is a delay of approximately 1 day in the division of cells heated to 42.0°, after which time some cells resume normal growth, whereas others divide at least once before death. Cells selected for their capacity to survive prolonged periods of hyperthermia are killed at approximately the same rate during subsequent heat treatments as cells that had not been heated previously. Hyperthermic cell killing is reduced in cells that are heated in the presence of certain compounds that are inhibitors of DNA and protein synthesis. Fractionated dose experiments indicate that cells recover from sublethal hyperthermic damage. Furthermore, hyperthermic killing is at least a two-step process, and cells are capable of recovery from potentially lethal damage, particularly in the presence of cycloheximide and high levels of thymidine.

Test for Carcinogenicity of Food Additives and Chemotherapeutic Agents by the Pulmonary Tumor Response in Strain A Mice

Abstract: Summary Forty-one food additives and 20 chemotherapeutic agents were examined for their ability to induce primary lung tumors in A mice. The animals received i.p. injections of each compound for 8 weeks and were killed at 24 weeks after the first injection. With some of the more toxic compounds, fewer injections were administered. Two or three dose levels were used for each chemical. Cinnamyl anthranilate was the only positive of the 41 food additives tested. Safrole and sodium saccharin were negative for pulmonary tumor response under the conditions used. Of the 20 chemotherapeutic agents, including alkylating chemicals, 12 were found to be positive in inducing pulmonary tumors. Uracil mustard was the most active compound. Myleran was inactive at the dose levels used.

Isolation of the hydrocarbon-deoxyribonucleoside products from the DNA of mouse embryo cells treated in culture with 7-methylbenz(a)anthracene-3H.

Abstract: Summary DNA from mouse embryo cells that had been treated in culture with 7-methylbenz[a]anthracene-3H was degraded with enzymes to deoxyribonucleosides, and deoxyribonucleoside-hydrocarbon products were separated from the normal deoxyribonucleosides by chromatography on Sephadex LH-20 columns eluted with water: methanol gradients. The 7-methylbenz[a]anthracene-3H-deoxyribonucleoside products were eluted as two peaks, the second of which appears to consist of at least two products. Only one product peak was released by treatment of the DNA with dilute acid. The material in both peaks that was released by enzyme digestion exhibited the thin-layer chromatographic behavior anticipated for hydrocarbon-deoxyribonucleoside products. Similar column elution profiles were obtained with enzyme digests of DNA isolated from mouse skin, mouse embryo cells in primary culture, and human embryo lung cells in primary culture following treatment with 7-methylbenz[a]anthracene-3H. In addition to the tritium present in the hydrocarbondeoxyribonucleoside products, a substantial portion of the tritium associated with the DNA from the above sources was found in the normal deoxyribonucleosides. The chromatographic system described separates the normal deoxyribonucleosides from the hydrocarbon-deoxyribonucleoside products and allows the actual amount of hydrocarbon bound to DNA to be determined.

Distribution of Kinase and Deaminase of 1-β-d-Arabinofuranosylcytosine in Tissues of Man and Mouse

Abstract: Summary 1-β-d-Arabinofuranosylcytosine (ara-C), an important antitumor agent, must be converted to the nucleotide by deoxycytidine kinase (K) in order to be biologically active It is inactivated by ara-C deaminase (D) to 1-β-d-arabinofuranosyluracil The enzymes are widely distributed in normal and neoplastic tissues of both man and mouse Low K and high D activities, thus low ratios of K to D, were demonstrated in chronic myelogenous leukemic cells and human normal peripheral white cells and marrow Acute myelogenous leukemic and chronic lymphocytic leukemic cells either from peripherals or marrows had relatively high K/D ratios A high K was demonstrated in spleens of man and mouse The human spleen and liver had high D, whereas mouse kidney had high D On the basis of equal body weight, human tissues contained more D than did mouse tissues This corresponds well with the faster catabolic rate of ara-C in man than in mouse in vivo In mice, the ip administration of a D inhibitor, tetrahydrouridine (100 mg/kg), decreased the amount of 1-β-d-arabinofuranosyluracil and proportionally increased the amount of ara-C excreted in urine In view of the higher D in man than in mouse, tetrahydrouridine in combination with ara-C may be of more value clinically than ara-C alone

Selective lethal effect of supranormal temperatures on mouse sarcoma cells.

Abstract: Summary The effects of supranormal temperatures upon normal (derived from normal, embryonal tissues) and neoplastic (derived from 3-methylcholanthrene-induced sarcomas) mesenchymal cells from C57BL/6 mice have been quantitatively studied in tissue culture. It has been found that exposure to a temperature of 42.5° for 2 hr or more has a selective lethal effect upon tumor cells (defined as cells capable of developing into a malignant tumor when injected into syngenic mice at doses of 1 × 10 6 cells or less in adults or 1 × 10 5 or less in newborns). No other biological characteristic studied (aneuploidy, length of time in culture, and rate of growth) could be correlated significantly with thermosensitivity. All the cultures of tumor-derived and tumor-producing cells exhibited the same high degree of thermosensitivity, i.e. , death of 95% of the cells after 2 hr exposure at 42.5°. All the cultured normal, nontumor-producing cells exhibited a lower degree of heat sensitivity, i.e. , death of 43% of the cells after 2 hr exposure to 42.5°. When a cell subline having a high tumor-producing ability was derived from a nontumor-producing line, it acquired a greater thermosensitivity, although its rate of growth was the same as that of the original line. These results indicate that the acquisition of biological malignant potential, both in vitro and in vivo , is accompanied by increased thermosensitivity in mouse mesenchymal cells.

Spontaneous Tumors in Sprague-Dawley Rats and Swiss Mice

Abstract: Summary A spontaneous tumor incidence of 45% was noted in 360 Sprague-Dawley rats (179 males and 181 females) and a 26% incidence was seen in 254 Swiss mice (101 males and 153 females) used as untreated control animals in an 18-month series of carcinogenesis experiments. The percentage of female rats with tumors was almost double that of males, which difference was accounted for chiefly by the high incidence of mammary tumors in the females. The largest number of rat tumors occurred in the endocrine system, mainly the pituitary and adrenal glands, with females exhibiting a higher incidence than males. There were no liver tumors. The largest group of mouse tumors occurred in the pulmonary system, with a higher incidence in females than in males. Urinary system tumors were observed in the males but not in the females. Tumors of the integument, reproductive organs, and the reticuloendothelial and lymphatic organs were also observed.

Effects of camptothecin on the breakage and repair of DNA during the cell cycle.

Abstract: Summary Incubation of camptothecin with HeLa cells in either the G1 or S phase of the cell cycle results in fragmentation of DNA as analyzed on alkaline sucrose gradients. Removal of the drug from G1- or S-phase cells results in the normal sedimentation of cellular DNA. Neither the fragmentation nor the repair of HeLa cell DNA requires DNA replication. However, subtle irreversible changes must take place in the DNA in cells treated with camptothecin for 30 min during the S phase, since such cells are unable to undergo subsequent cell division. The lethal effect is presumed to be at the single strand-replication forks.

A method for measuring DNA damage and repair in the liver in vivo.

Abstract: Summary Alkaline sucrose gradients were used to study the induction of single-strand breaks in and repair of rat liver DNA in vivo. Liver DNA was labeled during regeneration after partial hepatectomy. DNA was isolated from the organ in such a way as to minimize the induction of breaks due to handling or enzymatic activity. Different methods of preparing high-molecular-weight liver DNA were compared. Best results were obtained by squashing the liver in an ethylenediaminetetraacetic acid-sodium chloride buffer. Using this squash technique and alkaline sucrose gradients, high-molecular-weight DNA was regularly obtained. This DNA behaved like single-stranded DNA on hydroxyapatite columns and contained no detectable contamination of protein or RNA. It was demonstrated by the method described that hepatic DNA damage was produced within four hr by the administration of methyl methanesulfonate and that the rat can repair such hepatic DNA damage in vivo within 48 hr.

Improved therapeutic index of methotrexate with "leucovorin rescue".

Abstract: Summary The effect of “leucovorin rescue” on the therapeutic index of methotrexate in epidermoid carcinomas of the head and neck was studied. Sixteen patients received maximally tolerated doses of methotrexate alone as 30-hr i.v. infusions at 2-week intervals and 44% responded. Significant hematological toxicity and 4 drug-related deaths were incurred. Twenty-five patients received higher doses of methotrexate followed by leucovorin rescue as 36- to 42-hr i.v. infusions every 2 weeks. This group experienced a response rate of 60% with fewer cases of severe toxicity. Seventeen of this group received more than 500 mg/sq m as a maximal dose and had a response rate of 76% with two drug-related deaths, while those receiving smaller doses had a response rate of only 25%. Serum methotrexate levels were proportional to the dose of drug. Leucovorin rescue enhances the therapeutic index of high-dose methotrexate infusions in epidermoid carcinomas of the head and neck.

K-Region and Non-K-Region Metabolism of Benzo(a)pyrene by Rat Liver Microsomes

Abstract: Summary The metabolism of benzo(a)pyrene (BP) by liver microsomes from normal and 3-methylcholanthrene (MC)-treated rats was quantitatively analyzed by a double label method using BP-3H and BP-14C. Qualitatively, the metabolism of BP by both microsomal preparations was similar. The identified metabolites were 7,8-dihydro-7,8-dihydroxy-BP, 4,5-dihydro-4,5-dihydroxy-BP, 9,10-dihydro-9,10-dihydroxy-BP, 3-hydroxy-BP, 9-hydroxy-BP, and BP-1,6-quinone and BP-3,6-quinone. The quantitative analysis by the double labeling method showed the profile of BP metabolites produced by rat liver microsomes from normal and MC-induced rats. The ratio of metabolites from induced microsomes to that from normal microsomes was greatest for the 7,8-dihydro-7,8-dihydroxy and 9,10-dihydro-9,10-dihydroxy metabolites, less for the phenols and K-region metabolites, and least for the quinones. Similar ratios were obtained when microsomes from MC-treated rats were diluted except that dilution reduced the relative amount of phenol formation. These results suggest that higher enzyme activity favors the formation of the non-K-region diols and phenols relative to the K-region diol.

A Biologically Active Metabolite of Cyclophosphamide

Abstract: Summary N,N-Bis(2-chloroethyl)phosphorodiamidic acid has been isolated from incubations of cyclophosphamide with mouse liver microsomes. The compound was identified by mass spectrometry. N,N-Bis(2-chloroethyl)phosphorodiamidic acid has been synthesized and studied previously by Friedman and was found to be a potent alkylating agent with both in vivo and in vitro antitumor activity. This compound may play a major role in the biological activities of cyclophosphamide.

Patterns of Damage and Repair of Liver DNA Induced by Carcinogenic Methylating Agents in Vivo

Abstract: Single-strand breaks induced by methylating agents in liver DNA and their rejoining were studied in vivo by the use of alkaline sucrose gradients. DNA breaks were induced by the i.p. injection of single doses of dimethylnitrosamine (DMN), methylazoxymethanol acetate (MAM), N -methyl- N -nitrosourea (MNU), and N -methyl- N -nitrosourethan. The degree of damage was dependent on the dose of the substance given. DMN and MAM are comparably efficient in inducing marked single-strand breaks of liver DNA in small doses. MNU, although noncarcinogenic for liver, causes single-strand breaks of liver DNA, while N -methyl- N -nitrosourethan causes less damage even at toxic doses. The repair of the damage to DNA induced by MNU was complete in the first week while that induced by DMN and MAM was still not complete within 14 days after administration of the compond. Thus, the repair of the damage induced by hepatocarcinogens (DMN and MAM) seemed to be slower than that with the methylating agents not carcinogenic for the liver (MNU and methyl methanesulfonate).

1'-Hydroxysafrole, a proximate carcinogenic metabolite of safrole in the rat and mouse.

Abstract: Summary When fed as 0.5% of the diet for 8 to 10 months, 1′-hydroxysafrole induced a high incidence of hepatocellular carcinomas in male rats; safrole induced only a low incidence under these conditions. 1′-Acetoxysafrole, because of its toxicity, was fed to rats at only 0.6 the molar level of the above compounds. These rats did not develop hepatic tumors, but all of those that survived at least 6 months developed multiple papillomas of the forestomach; squamous cell carcinomas were found in the forestomachs of two of these rats. A few isolated papillomas of the forestomach were observed in rats fed 1′-hydroxysafrole. Eighty-four and 82%, respectively, of male mice given injections of 1′-hydroxy- or 1′-acetoxysafrole at 1 to 21 days of age (total dose, 9.5 µ;moles) and killed at 12 to 14 months of age had liver tumors; the incidences were 40% for male mice given injections of safrole and 8% for the controls. Of the adult male mice fed 0.4 or 0.5% of safrole for 13 months and killed at 16 months, 30% had liver tumors; the incidence was 11% in the controls. Some liver tumors also developed in adult male mice fed 1′-hydroxysafrole, but the number of survivors was low. All of these liver tumors were diagnosed as highly differentiated hepatic carcinomas. Between the 14th and 16th months, 46% of the mice that were fed 1′-hydroxysafrole and that survived at least 12 months developed sarcomas, most of which were diagnosed as angiosarcomas, in the interscapular region; only two safrole-fed mice and one control mouse developed tumors at this site. Twenty s.c. injections of 18.6 µmoles each of 1′-acetoxysafrole or 1′-hydroxysafrole induced sarcomas in 30 and 8%, respectively, of adult male rats. These sarcomas were observed 12 to 18 months after the 1st injection. Skin tumors did not develop in mice treated topically 14 times with 1.8 µmoles of safrole; its 1′-hydroxy, 1′-methoxy, or 1′-acetoxy derivatives; or the corresponding 2′,3′-dihydrosafrole derivatives and then given twice weekly applications of phorbol-12,13-didecanoate. On the basis of the above data and our finding that a conjugate of 1′-hydroxysafrole is excreted in the urine of rats and mice given p.o. or i.p. safrole, we conclude that 1′-hydroxysafrole is a proximate carcinogenic metabolite of safrole.