Showing papers in "Cancer Research in 1978"

Photoradiation therapy for the treatment of malignant tumors

Abstract: Administration of hematoporphyrin derivative i.v. followed by local exposure to red light has resulted in complete or partial response in 111 of 113 cutaneous or s.c. malignant lesions. Tumors treated have included carcinomas of the breast, colon, prostate, squamous cell, basal cell, and endometrium; malignant melanoma; mycosis fungoides; chondrosarcoma; and angiosarcoma. No type has been found to be unresponsive. In several cases complete clearing of chest wall metastatis has been achieved in treated areas. Deep-seated and pigmented tumors required a higher dose of drug for effective treatment than did the more superficial and nonpigmented lesions. A high therapeutic ratio between tumor and skin response has been obtained by allowing at least 3 days between drug injection and exposure to the therapeutic light for 2,5-mg/kg doses and at least a 4-day interval for 5.0-mg/kg doses.

Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones.

Abstract: Continuously cultured human neuroblastoma cell lines SK-N-SH, SK-N-BE(1), SK-N-BE(2), and SK-N-MC, as well

Tumor heterogeneity and the biology of cancer invasion and metastasis.

Abstract: The development of a metastasis is dependent on an interplay between host factors and intrinsic characteristics of malignant tumor cells. The process of metastasis is highly selective, and the metastatic lesion represents the end point of many destructive events that only a few cells can survive. Neoplasms, which are predominantly heterogeneous, contain a variety of subpopulations of cells with differing metastatic potential. Furthermore, metastatic cell variants have been shown to preexist in murine neoplasms of old and recent origin. The possible existence of highly metastatic variant cells within a primary tumor suggests that we no longer should consider a neoplasm to be a uniform entity. Efforts to design effective therapeutic agents and procedures against malignant tumors should be directed toward the few but fatal metastatic subpopulations of cells.

A general mechanism for microsomal activation of quinone anticancer agents to free radicals.

Abstract: The highly active, quinone-containing anticancer drugs, Adriamycin, daunorubicin, carminomycin, rubidazone, nogalamycin, aclacinomycin A, and steffimycin (benzanthraquinones); mitomycin C and streptonigrin (N-heterocyclic quinones); and lapachol (naphthoquinone) interact with mammalian microsomes and function as free radical carriers. These quinone drugs augment the flow of electrons from reduced nicotinamide adenine dinucleotide phosphate to molecular oxygen as measured by enhanced reduced nicotinamide adenine dinucleotide phosphate oxidation and oxygen consumption. This reaction is catalyzed by microsomal protein and produces a free radical intermediate form of the drugs as determined by electron paramagnetic resonance spectroscopy. Microsomes from mouse and rat liver, heart, lung, and spleen and mouse L1210 and P388 tumors all catalyze the augmented oxygen consumption. Apparent Km values determined with normal rat liver microsomes range from 0.49 × 10-4m for steffimycin to 13.4 × 10-4m for lapachol. Since SKF 525A and carbon monoxide have little effect on this reaction, cytochrome P-450 is probably not involved. Several nonquinone anticancer agents were tested and were found inactive in the system. Since quinone anticancer drugs are associated with chromosomal damage that appears to be dependent on metabolic activation of these drugs, we propose that the intracellular activation of these drugs to a free radical state may be primary to their cytotoxic activity. As free radicals, these drugs, because of their high affinity and selective binding to nucleic acids, have the potential to be “site-specific free radicals” that bind to DNA or RNA and either react directly or generate oxygen-dependent free radicals such as superoxide radical or hydroxyl radical to cause the damage associated with their cytotoxic actions.

Heterogeneity of tumor cells from a single mouse mammary tumor.

Abstract: By the use of a variety of cell culture and separation methods, four cell lines were isolated from a single autochthonous BALB/cfC3H mammary tumor. These lines differ markedly from each other in culture morphology, various in vitro growth properties, expression of murine mammary tumor virus antigen, and karyotype, yet all four lines are tumorigenic in normal, syngeneic hosts, yielding tumors of generally similar histology, although distinct from the original neoplasm. Three of the four lines have been cloned from soft agar. The clones exhibit the same growth properties as the lines from which they were derived. Karyotypic analysis of the parent tumor revealed the presence of cells with heterogeneous numbers of chromosomes similar to those seen in the isolated lines, suggesting both the presence of these distinct cell types in the original neoplasm and a genetic origin of the diversity.

A clinical-pharmacological evaluation of hepatic arterial infusions of 5-fluoro-2'-deoxyuridine and 5-fluorouracil.

Abstract: We have attempted to evaluate the degree to which hepatic arterial infusion of 5-fluoro-2′-deoxyuridine (FdUrd) or 5-fluorouracil produces higher hepatic and lower systemic drug concentrations than are achieved with corresponding peripheral venous infusions. Hepatic arterial catheters were placed for therapy in 15 patients with primary or metastatic liver cancer. Temporary hepatic venous catheters allowed direct sampling of drug levels in the hepatic venous effluent as well as measurement of hepatic blood flow. FdUrd was measured primarily by radioimmunoassay and fluorouracil by a high-pressure liquid chromatographic system. Due to the limited sensitivities of these assays, short (40 to 60 min) infusions at dose rates 10 to 100 times those conventionally used were given in order to produce drug levels that could be measured reliably. Even at dose rates of 0.5 to 40 mg/kg of body weight/hr for FdUrd and 5.6 mg/kg of body weight/hr for fluorouracil, steady state drug levels were achieved in the bloodstream in 30 to 40 min and hepatic extraction could be quantified. The hepatic extraction of FdUrd is high with an extraction ratio (hepatic arterial level — hepatic venous level/hepatic arterial level) of 0.69 to 0.92 and a clearance of 0.81 to 2.3 liters/min. The hepatic extraction of fluorouracil, however, appears to be lower with an extraction ratio of 0.22 to 0.45 and a clearance of 0.24 to 0.45 liters/min. With hepatic arterial drug infusion, 94 to 99% of FdUrd and 19 to 51% of fluorouracil is extracted in one pass. Hepatic venous levels, which are one measure of intrahepatic drug concentration in the hepatic and tumor capillary bed, were 4-fold higher for FdUrd infusion and 1.5-fold higher for fluorouracil infusion when drug was given by the hepatic arterial route. Systemic FdUrd levels with hepatic arterial infusion were only about 25% of corresponding systemic levels with peripheral venous infusion. Systemic fluorouracil levels with hepatic arterial infusion were also lower and were about 60% of corresponding systemic levels with peripheral venous infusion. These results support hepatic arterial infusion as a means to improve the therapeutic index of FdUrd and fluorouracil in the treatment of cancer in the liver. Although molar equivalent dose rates of FdUrd and fluorouracil were used in this study, the differences in FdUrd and fluorouracil pharmacology as noted above may not be applicable to conventional hepatic arterial therapy where much lower dose rates are used. Nonetheless, this type of analytical approach should prove valuable in the evaluation of other agents for hepatic arterial chemotherapy.

Determination of Cellular Shape by the Extracellular Matrix and Its Correlation with the Control of Cellular Growth

Abstract: Although the problem of cellular proliferation may seem at first glance to be tremendously complex, the mechanisms which control it may be extremely simple One of the primary factors which regulates the mitogenic response of a given cell type to a given class of mitogenic agents seems to be the cellular shape We have found that corneal epithelial cells, for example, adopt a flattened configuration when maintained in vitro on plastic and are very sensitive to fibroblast growth factor, but not to epidermal growth factor When maintained on collagen, on the other hand, they become tall and columnar and respond primarily to epidermal growth factor The cellular shape is dictated in vitro by the extracellular material upon which the cells rest and in vitro by the substrate upon which the cells are maintained The substrate itself may, in turn, induce the cells to manufacture their extracellular material and specific cell surface proteins which control the cellular shape

Inhibition of Polycyclic Aromatic Hydrocarbon-induced Neoplasia by Naturally Occurring Indoles

Abstract: Indole-3-carbinol, 3,3'-diindolylmethane, and indole-3-acetonitrile, three indoles occurring in edible cruciferous vegetables, have been studied for their effects on 7,12-dimethylbenz(a)anthracene-induced mammary tumor formation in female Sprague-Dawley rats and on benzo(a)pyrene-induced neoplasia of the forestomach in female ICR/Ha mice. When given by p.o. intubation 20 hr prior to 7,12-dimethylbenz(a)anthracene administration, indole-3-carbinol and 3,3'-diindolylmethane had an inhibitory effect on mammary tumor formation, but indole-3-acetonitrile was inactive. Indole-3-carbinol when added to the diet for 8 days prior to challenge with 7,12-dimethylbenz(a)anthracene inhibited mammary tumor formation, whereas indole-3-acetonitrile did not. Dietary administration of all three indoles inhibited benzo(a)pyrene-induced neoplasia of the forestomach in ICR/Ha mice. The identification of dietary constituents that can inhibit chemical carcinogens ultimately may be of value in understanding the balance of factors that determines the neoplastic response to these cancer-producing agents in the environment.

Establishment and Characterization of Three New Continuous Cell Lines Derived from Human Breast Carcinomas

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

Heat Fractionation and Thermotolerance: A Review

Abstract: A rational approach to the design of clinical protocols combining fractionated hyperthermia plus X-Irradiation or hyperthermia plus chemotherapy requires an understanding of the biology of fractionated heat alone. Mammalian cells growing in vitro can dramatically increase their tolerance to thermal damage ( i.e. , reduce the cellular inactivation rate) after prior heat conditioning. Although the mechanism(s) for this cellular thermotolerance is still unknown, it is apparent that the thermal history, the heat fractionation interval, and the recovery conditions all modify significantly the degree of thermotolerance subsequently exhibited. At the tissue level, the role of cellular thermotolerance is further complicated by host physiological mechanisms. Few data are available on heat fractionation in vivo , and the relative importance of physiological versus cellular effects remains to be defined.

Human breast carcinoma cells in continuous culture: a review.

Abstract: A comprehensive listing of putative human breast carcinoma cell lines and the extent to which each has been characterized is presented. Criteria used to certify the human, mammary, and malignant origin of a cell line include: ( a ) a reliable histopathological diagnosis; ( b ) interspecies specificity established by human karyotype, isoenzyme profiles, and/or cell surface antigenicity; ( c ) intraspecies specificity, demonstrated by genetic evidence of a unique, human donor distinct from other cells including HeLa cells; and ( d ) organ specificity, supported by morphological evidence of epithelial structure and secretory activity, and especially by the expression of differentiated functions; these include presence of receptors for sex steroid hormones, hormone responsiveness, and production of milk proteins, fatty acids, or milk-specific antigens. Of the 47 cell lines for which data are here reported, 22 have been shown to be derived from human, non-HeLa donors and to have epithelial morphology as revealed by light or electron microscopy. Differentiated function has been recorded for 19 cell lines. Additional human breast cancer cell lines have been reported, but characterization of some of these has been insufficient to judge the legitimacy of their pedigrees. For others mammary origin is questionable. Six purported breast cell lines are in reality HeLa cells, and one is of nonhuman origin.

Elevation of Hepatic Glutathione S-Transferase Activities and Protection against Mutagenic Metabolites of Benzo(a)pyrene by Dietary Antioxidants

Abstract: Addition of either 2(3)-tert-butyl-4-hydroxyanisole (BHA) or 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline (ethoxyquin) to the diet greatly decreases the levels of mutagenic metabolites of benzo(a)pyrene in CD-1 mice. (R. P. Batzinger, S-Y. L. Ou, and E. Bueding, Cancer Res., 38:000, 1978). The mutagenic activity of the urinary metabolites of benzo(a)pyrene is markedly reduced in the presence of glutathione together with the liver cytosols of rats or mice fed on a diet containing BHA. The liver cytosols of mice and rats maintained on control diets are much less effective in this respect. Dietary BHA causes increases in mouse and rat hepatic glutathione S-transferase (EC 2.5.1.18) specific activities with 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, p-nitrobenzylchloride, and Δ5-androstene-3,17-dione. In the mouse the increases are larger (5- to 10-fold) and are dependent on the dose and duration of administration of BHA. Increases in these glutathione S-transferase specific activities were also observed in mouse hepatic cytosols after feeding of ethoxyquin. Direct addition of reduced glutathione and purified glutathione S-transferases A and B obtained from rat liver to the mutagenicity assay system mimicked the effect of the rodent cytosols. Since BHA and ethoxyquin are known to reduce the neoplastic effects of a variety of potent carcinogens, we suggest that the protective effects of these antioxidants may be accounted for, at least in part, by their ability to elevate the glutathione S-transferases. These enzymes inactivate arene oxides and other hydrophobic electrophiles by catalyzing their conjugation with glutathione.

Steroid receptor analyses of nine human breast cancer cell lines.

Abstract: Nine human breast cancer cell lines in permanent tissue culture and currently available to researchers have been assayed for their content of cytoplasmic estrogen receptors, progesterone receptors, androgen receptors, and glucocorticoid receptors, as well as for the presence of unfilled or hormone-filled nuclear estrogen receptors. Receptor distribution varied considerably among the nine lines and differed from the expected distribution predicted from solid tumors. We find that estrogen receptor, when present, is usually localized in the nucleus as unfilled nuclear estrogen receptor. Progesterone receptor is correlated with presence of unfilled nuclear estrogen receptor. Glucocorticoid receptors are ubiquitous; they were found in all cell lines tested. The distribution of androgen receptor and progesterone receptor differed, suggesting that these proteins are dissimilar.

Direct Cloning of Human Ovarian Carcinoma Cells in Agar

Abstract: We have recently developed an in vitro assay for human tumor stem cells that permits cloning of human ovarian adenocarcinoma cells in soft agar. Tumor colonies grew from both effusions and biopsies from 85% of 31 ovarian cancer patients. The cloning efficiency did not vary with the histology of the tumor. Growth was induced with medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. Up to 2000 colonies appeared after 10 to 14 days in culture, yielding a plating efficiency of 0.001 to 1%. Cells from nonmalignant effusions did not form colonies under these conditions. The number of tumor colonies was proportional to the number of cells plated between concentrations of 104 to 106 cells/dish. Morphological and histochemical criteria showed that the colonies consisted of cells with the same characteristics as those of the original tumor. Results of cytogenic studies were also consistent with a malignant origin for the tumor colonies with marked hyperdiploidy in colonies from four patients and hypodiploidy in a fifth patient. [3H]Thymidine and hydroxyurea suicide indices provided evidence that in most cases a high proportion of ovarian tumor colony-forming cells were actively in transit through the cell cycle. Removal of phagocytic macrophages with carbonyl iron markedly reduced the plating efficiency, and 2-mercaptoethanol could only partially substitute for macrophages. The assay appears useful for screening differential cytotoxic effects of specific anticancer drugs (such as cis -platinum) against the tumor stem cells from various ovarian cancer patients.

Heterogeneity in Drug Sensitivity among Tumor Cell Subpopulations of a Single Mammary Tumor

Abstract: Three distinct subpopulations of tumor cells derived from a single parent strain BALB/cfC3H mammary adenocarcinoma were tested in vivo for sensitivity to cyclophosphamide, methotrexate, and 5-fluorouracil. Treatment was begun either 2 days after s.c. tumor cell injection or at the time when the tumors became palpable. It was given on a weekly basis for 4 weeks. The mice were observed for growth of the primary implant and for development of spontaneous metastases. The three subpopulations differed markedly in their sensitivity to the drugs. The effects of the drugs ranged from induction of regression of the "primary" to enhancement of metastases. The effect on primary growth was independent of that on metastasis. The effect of the time of administration of the drugs also varied among the subpopulations. The sublines were also tested in vitro with methotrexate and 5-fluorouracil. Again there were marked differences in sensitivity to inhibition of cell division by the drugs. The relative sensitivities in vitro did not correlate with observations in vivo. The existence of subpopulations of tumor cells, differing in sensitivity to therapeutic agents, within a single neoplasm, presents a challenge to development of assays capable of predicting drug response and to the selection of combination therapies.

Mechanisms of Resistance to Daunorubicin in Ehrlich Ascites Tumor Cells

Abstract: Uptake and binding of daunorubicin were studied in a sensitive (EHR 2) and in a resistant (EHR 2/DNR+) subline of Ehrlich ascites tumor cells. At steady state, the cell:medium ratio of daunorubicin was about 10-fold higher in EHR 2 than in EHR 2/DNR+. The bindings of daunorubicin to cell homogenate of the two cell lines were equal when the concentration at equilibrium was below 0.7 µg/ml, whereas homogenate of EHR 2 bound significantly more daunorubicin at higher concentrations. Based on the binding affinity for cell homogenate, the cytoplasm:medium ratio at steady state was estimated to be below 0.15 in EHR 2/DNR+, while the ratio in EHR 2 was 0.6 to 1.0. Omission of glucose together with the addition of sodium azide resulted in a considerable increase in drug uptake and in an equalization of the cytoplasm: medium gradient in EHR 2/DNR+. If glycolysis was restored by the addition of glucose to cells treated with sodium azide and loaded with daunorubicin, and uphill exodus of the drug was induced in EHR 2/DNR+. In both cell lines omission of glucose together with sodium azide increased the initial rate of uptake. However, the increment was three times as high for EHR 2/DNR+ as for EHR 2. In the medium without glucose but with sodium azide, the influx followed simple saturation kinetics in both cell lines, indicating carrier-mediated transport. As a consequence of a lower Vmax, the influx was significantly lower in EHR 2/DNR+ than in EHR 2. The data indicate that lower drug uptake in cells resistant to daunorubicin may be a result of at least three different mechanisms: a lower influx, a higher active extrusion, and a lower affinity for intracellular binding sites.

Effects of dimethylnitrosamine on induction of cholangiocarcinoma in Opisthorchis viverrini-infected Syrian golden hamsters.

Abstract: Opisthorchiasis is an endemic parasitic disease in northeastern Thailand. The concomitant occurrence of the parasite and cholangiocarcinoma as well as the incidence of such tumors are higher in that part of the country than in other areas. Nitrosating agents, nitrosatable substances, and nitrosamine compounds are commonly present in several kinds of stable foods in that region. To study the interactions between the parasite and the nitroso compounds, we divided Syrian golden hamsters into four groups: Group 1, untreated; Group 2, dimethylnitrosamine (DMN, 0.0025% or 25 ppm)-treated; Group 3, 100 metacercariae-treated; and Group 4, DMN (0.0025% or 25 ppm) plus 100 metacercariae-treated groups. The animals that received both DMN and parasites (Group 4) developed cholangiocarcinoma (100%) and cholangiofibrosis (100%). The tumor was not observed in the group that received either DMN (Group 2) or parasites alone (Group 3), although cholangiofibrosis was found in some animals in the DMN group (Group 2). It is postulated that cholangiocarcinomas in these animals arose because the DMN exerted a carcinogenic effect on the altered proliferating epithelial cells of bile ducts that had been stimulated by the parasite. These findings suggest that the combination of DMN ingestion and liver fluke infestation may play an important role in the carcinogenesis of the intrahepatic duct neoplasms in human beings.

Mechanism of cross-resistance between vincristine and daunorubicin in ehrlich ascites tumor cells.

Abstract: An investigation was undertaken of the mechanism of a previously reported cross-resistance between vincristine (VCR) and daunorubicin (DNR) in Ehrlich ascites tumor cells. No significant difference was demonstrated for the time course of [3H]VCR uptake in cells resistant to VCR (EHR 2/VCR+) and in cells resistant to DNR (EHR 2/DNR+), whereas wild-type cells accumulated nearly 6-fold more drug at steady state. The energy dependence of [3H]VCR and of DNR transport was investigated by the metabolic inhibitors sodium azide and iodoacetic acid. These studies revealed that uptake of [3H]VCR and of DNR was depressed in both resistant sublines by an energy-dependent process that mostly requires energy from glycolysis. If glucose was omitted from the medium together with addition of sodium azide, the uptake of [3H]VCR and of DNR in EHR 2/VCR+ reached a level nearly equal to that of wild-type cells. If glycolysis was restored by addition of glucose to the resistant cells loaded with drug in this way, a pronounced extrusion of [3H]VCR and of DNR was induced. In a similar experiment with wild-type cells, a slight but significant extrusion of [3H]VCR could be induced. The studies showed that, for nearly unidirectional influx, the cells must be incubated in the medium without glucose but with sodium azide. In this medium the influx of [3H]VCR and of DNR was significantly higher in wild-type cells than in cells from the resistant sublines. The flux of DNR was not competitively inhibited by VCR either in wild-type cells or in resistant cells. The data indicate that the mechanism of cross-resistance between VCR and DNR in Ehrlich ascites tumor cells is a result of at least two different mechanisms: (a) an energy-dependent drug extrusion common to VCR and DNR; and (b) unspecific changes in the membrane, which reduce the influx of both compounds.

Effects of Hyperthermia on Survival and Progression of Chinese Hamster Ovary Cells

Abstract: In general, the survival of Chinese hamster ovary cells exposed to hyperthermic temperatures of 42.5–46.0° decreases exponentially as a function of duration of heat exposure in a manner quite similar to survival as a function of radiation dose. The data indicate that above 43° a 1° change in temperature requires a 2-fold change in time to achieve the same degree of killing, whereas below 43° the same 2-fold change in time requires only a 0.5° change in temperature for the same effect. An Arrhenius-type plot of the logarithm of the rate of killing as a function of reciprocal temperature exhibits linearity with a change in slope at 43°. This change in slope suggests either a change in the mechanism of cell killing below this temperature or a manifestation of thermal tolerance that is readily observed when the duration of heating exceeds 4 to 5 hr. Thermotolerance to 45.5°, as evidenced by a 3- to 4-fold increase in D0, is observed in synchronous G1 cells exposed to heat 20 hr after an initial heat dose. This thermotolerance develops, although no progression of cells into S phase occurs during this period. In addition, thermotolerance develops in both asynchronous and synchronous G1 cells exposed to single heat doses between 41.5 and 42.5° for periods exceeding 4 to 5 hr, i.e., survival decreases exponentially as a function of duration of heating up to 4 to 5 hr, after which survival decreases very little. At 42.0–42.5°, survival is extremely sensitive to changes in temperature, with as much as a 10-fold difference in survival for a 0.1° difference in temperature with heat exposures greater than 4 hr. The above data indicate the importance of careful treatment design and precise temperature control if hyperthermia is to be used for cancer therapy. No progression of synchronous G1 cells into S phase is observed for cells continuously exposed to temperatures of 42.0° and above. However, computer simulation of sequential DNA histograms from flow cytometry of synchronous cells continuously exposed to 41.5° indicates that cell cycle delays of 5.4 and 2.4 hr for G1 and S, respectively, occurred for cells for which exposure began in G1, and delays of 0.5 and 5.4 hr for S and G2 plus mitosis, respectively, occurred for cells for which exposure began in late S. Normal cell cycle phase transit times for G1, S, and G2 plus mitosis are 4.3, 7.1, and 2.4 hr, respectively. In addition, mitotic indices and increases in cell number of asynchronous populations of cells continuously exposed to 41.5° indicate that entry into mitosis is delayed for approximately 2 hr. Following this delay, cells begin to enter mitosis and accumulate from 2 to 6 hr in metaphase; after about 6 hr, they begin to progress into G1. However, comparison of flow cytometry data and mitotic index data suggests that during the initial 6 hr of heating, the majority of cells accumulating in G2 plus mitosis are actually delayed in G2.

Early Histological and Functional Alterations of Ethionine Liver Carcinogenesis in Rats Fed a Choline-deficient Diet

Abstract: The effects of feeding a choline-deficient (CD) or a choline-supplemented diet upon the early stages of DL-ethionine carcinogenesis in rat liver were investigated. Low levels of DL-ethionine (0.05 and 0.10%) when fed with a CD diet were found to induce within 4 weeks a massive proliferation of oval cells without significant cell necrosis or presence of inflammatory cell infiltrates. The same levels of ethionine when fed with a choline-supplemented diet caused no significant histological alteration of the liver. In rats fed the CD plus ethionine diets concomitant with the proliferation of oval cells, there was a marked elevation in the content of alpha1-fetoprotein in both liver and plasma. After specific immunofluorescence staining, oval cells stained intensely for albumin and alpha1-fetoprotein. Hepatocytes stained only for albumin, and bile duct cells stained for neither albumin nor alpha1-fetoprotein. These results indicate that a diet deficient in choline markedly alters the response of rat liver to carcinogenetic doses of ethionine. Thus, ethionine hepatocarcinogenesis in rats fed a CD diet may be a useful model for the exploration of the mechanism(s) whereby a dietary factor influences hepatocarcinogenesis.

Correlation of Anchorage-independent Growth with Tumorigenicity of Chemically Transformed Mouse Epidermal Cells

Abstract: The BALB/c mouse primary epidermal cell culture system is currently being developed as a model system for chemical carcinogenesis studies with the immediate aims of achieving a high incidence of chemically induced transformation and developing rapid assays for tumorigenicity Primary cultures treated with N-methyl-N′-nitro-N-nitrosoguanidine (1 to 2 µg/ml) at 0 to 3 days postplating, followed in some cases by phorbol ester treatment, yielded after 3 to 4 months six morphologically altered long-term cell strains of which three were tumorigenic, as determined by injection into syngeneic newborns One phorbol ester control yielded a tumorigenic strain while two solvent controls gave rise to long-term strains that were nonmalignant All strains examined showed the presence of intercellular intermediate junctions, an epithelial marker No morphological feature distinguished tumorigenic from nontumorigenic strains Growth rate in monolayer culture and growth in semisolid medium were investigated as potential correlates of tumorigenicity in 16 morphologically altered epidermal cell strains Although the tumorigenic cell strains showed a trend toward shorter doubling times, rapid growth in monolayer culture was not a consistent correlate of tumorigenicity In contrast, colony formation in 033% agar medium consistently correlated with tumorigenicity, with all tumorigenic strains positive and all nontumorigenic strains negative Colonyforming efficiency in soft agar, a parameter that was influenced by initial cell density and serum concentration, did not consistently parallel the degree of tumorigenicity

Metastatic Heterogeneity of Cells from an Ultraviolet Light-induced Murine Fibrosarcoma of Recent Origin

Abstract: We wished to determine whether cells within a murine tumor of recent origin vary in their metastatic potential. A fibrosarcoma induced in a C3H- mouse by chronic ultraviolet irradiation was established in tissue culture, and 21 clones were produced from the parent line. For determination of whether cells with different metastatic potential preexisted in the population of whether the process of metastasis resulted from the random survival of cells with equal metastatic capabilities, the parent line and the 21 clones were tested for metastatic behavior in syngeneic mice. Three different in vivo tests were used: (a) mice were given injections of 105 tumor cells i.v., they were killed 18 days later, and the tumor colonies in the lungs were counted; (b) mice were given s.c. injections of 105 tumor cells and examined at death for the presence of spontaneous metastases; (c) mice were given i.v. injections of 105 tumor cells and autopsied at death. The time of death was recorded, and the number and site of metastases were determined. The clones varied greatly in their ability to grow and metastasize after s.c. or i.v. inoculation. The three tests gave similar results in that clones judged to be of high or low metastatic potential in one test usually exhibited the same behavior in the other two tests. We conclude that the parent tumor is heterogeneous and that cells with widely different metastatic potential preexist in the parental population.

Testing of Known Carcinogens and Noncarcinogens for Their Ability to Induce Unscheduled DNA Synthesis in HeLa Cells

Abstract: The ability of 51 compounds, of known carcinogenic potential, to induce “unscheduled DNA synthesis” in HeLa cells has been tested in the presence or absence of a rat liver mixed-function oxidase preparation. Chemicals tested included those giving erroneous results in bacterial mutagenicity assays as well as representative compounds from various classes of chemical carcinogens including nitrosamines, polycyclic aromatic hydrocarbons, aromatic amines, and mycotoxins. Of the compounds assayed, all noncarcinogens failed to induce DNA repair; of 38 compounds of demonstrated carcinogenicity, 34 were active; safrole, N -propyl- N -nitrosourea, aflatoxin B 2 and N -butyl- N -nitrosourea were, however, inactive. Six compounds for which carcinogenicity data are incomplete were active, namely, 4-nitro- o -phenylenediamine, 2-nitro- p -phenylenediamine, formaldehyde, 2,2′-dichlorobenzidine, 3,3′,5,5′-tetrafluorobenzidine, and 3,3′,5,5′-tetrachlorobenzidine. Three carcinogens that are weakly active or inactive in bacterial mutagenicity assays, i.e. , urethan, N -dimethyl- p -aminoazobenzene, and diethylstilbestrol were active in our assay. The bacterial mutagens sodium azide and 9-aminoacridine were both inactive. The use of this assay in a tier scheme for the short-term testing of potential chemical carcinogens is discussed.

Tumorigenicity studies with diol-epoxides of benzo(a)pyrene which indicate that (+/-)-trans-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene is an ultimate carcinogen in newborn mice.

Abstract: The tumorigenic activities of benzo(a)pyrene(BP), (+/-)-trans-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxide 1), (+/-)-trans-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxide 2), (+/-)-trans-7,8,-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP 7,8-dihydrodiol), and the tetraols derived from the hydrolysis of diol-epoxide 2 were evaluated in newborn mice. The mice were given injections sequentially of 4, 8, and 16 nmoles of each compound on the first, eighth, and fifteenth days of life, and the animals were killed when they were 28 weeks old. Diol-epoxide 1 was highly toxic in newborn mice, and most of the animals treated with this compound died before weaning. Diol-epoxide 2 and BP 7,8-dihydrodiol were, respectively, about 40- and 15-fold more active than BP in causing pulmonary adenomas. Vehicle-treated control animals had an average of 0.13 lung adenoma/mouse, whereas animals treated with BP, BP 7,8-dihydrodiol, or diol-epoxide 2 had, respectively, 0.24, 1.77 and 4.42 pulmonary adenomas/mouse. Diol-epoxide 1 and the tetraols derived from diol-epoxide 2 did not induce pulmonary adenomas. The inactivity of diol-epoxide 1 under the conditions of our study should be interpreted with caution because of the high toxicity of this compound. The results of our study provide evidence that BP 7,8-dihydrodiol is a proximate carcinogenic metabolite and that diol-epoxide 2 is an ultimate carcinogenic metabolite of BP in the newborn mouse.

Energy Balance and Body Composition in Cancer Patients

Abstract: Energy balance and body composition were studied in 10 cancer patients to investigate the interrelationship between energy expenditure and energy intake in the development of cancer cachexia. These data were compared to the findings in control patients of similar age with diseases affecting physical activities to about the same extent. The measurements of energy expenditure and intake were repeated when possible during progression of the malignant disease. Body composition in a larger group of cancer patients (n = 29) was compared to data from a group of healthy subjects within the same age range (n = 164). The cancer patients were below normal in body weight and body cell mass. Body fat was reduced in women with cancer. Energy intake (mean, 1270 kcal/day), although varying greatly, was not significantly different from the intake in the controls (mean, 1470 kcal/day). Both the daily energy expenditure (mean, 2020 kcal/24 hr) and the resting metabolic rate (mean, 1630 kcal/24 hr) were significantly greater in the cancer patients than they were in the controls (mean, 1420 and 1170 kcal/24 hr, respectively). Parallel to exacerbation of the disease and reduced energy intake, the energy expenditure and the resting metabolic rate increased in relation to body cell mass in two patients. After curative surgery both intake and expenditure of energy returned to normal levels in one patients. For a more accurate interpretation of the higher daily energy expenditure and resting metabolic rate found in the cancer patients, the influences of body weight, body cell mass, and height were ruled out. The results suggest that one of the reasons for the development of cancer cachexia is an increasing resting metabolic rate and daily energy expenditure.

Determination of Ploidy and Proliferative Characteristics of Human Solid Tumors by Pulse Cytophotometry

Abstract: Cellular DNA content is a discriminator of cell cycle stage and ploidy. Using pepsin digestion for cell dispersal, a combination of ethidium bromide and mithramycin for DNA fluorochromation, and a new sheath flow chamber in a PHYWE ICP-11 pulse cytophotometer, we obtained DNA histograms with high resolution (coefficient of variation, 2.0 to 7.0; mean, 3.3%) on biopsy samples of solid tumors. There were 78 observations in 26 patients (malignant melanoma, 7; breast carcinoma, 4; lung carcinoma, 3; lymphoma, 3; miscellanceous tumors, 9). Ploidy identification was conducted by mixing tumor cells with human granulocytes. The ratio of peak channel numbers for the G1/Q compartment of tumor cells to that of normal cells was termed the DNA index. All but one patient with multiple myeloma presented unimodal tumor cell DNA distributions. Except for one breast and one colonic carcinoma, all tumors had aneuploid DNA contents. Three patients presented with hypodiploid abnormalities, and the remainder showed varying degrees of hyperdiploidy (DNA index ranged from 1.07 to 2.40 with a mean of 1.60). Among the patients in whom serial DNA distribution analyses were conducted, only one showed an increase in DNA index from 1.08 to 1.52. This patient presented with immunoblastic lymphadenopathy and later progressed into immunoblastic sarcoma. Pretreatment cycle stagerelated DNA distribution patterns varied considerably for the entire patient population and for each diagnostic subgroup. No correlation between ploidy and proliferative cell characteristics was apparent.

Steroid Receptors in Human Breast Cancer

Abstract: The measurement of cytoplasmic estrogen receptor in tumors from patients with breast cancer is now well established Potential uses include prognosis of early recurrence following mastectomy, stratifying patients for adjuvant therapies, and selecting or rejecting endocrine therapy in advanced breast cancer The use of progesterone receptor measurements to improve our selection process has a good theoretical basis, and early reports now emerging indicate that the presence of both estrogen and progesterone receptor in a breast tumor predicts a high response rate to endocrine therapy Further work in this area is required, however, since patients with estrogen receptor but not progesterone receptor still have an appreciable response rate

Specificity of Arrest, Survival, and Growth of Selected Metastatic Variant Cell Lines

Abstract: Animal tumor models for blood-borne metastasis have been developed by in vitro cloning or in vivo selection of malignant tumor cell populations to obtain organ-preferring variant tumor cell lines with altered arrest, survival, invasion, and growth properties. Selection and some tumor cell characteristics of lung-, brain-, and ovary-colonizing metastatic B16 melanoma, liver-colonizing RAW114 lymphosarcoma, and lung-colonizing MSV3T3 vasoformative sarcoma variant lines will be discussed along with additional data, suggesting that tumor cells of varying malignant potential preeexist in the unselected tumor population.

Transport and metabolism of deoxycytidine and 1-beta-D-arabinofuranosylcytosine into cultured Novikoff rat hepatoma cells, relationship to phosphorylation, and regulation of triphosphate synthesis.

Abstract: The zero-trans transport of deoxycytidine and 1-β-d-arabinofuranosylcytosine was determined in cultured Novikoff rat hepatoma cells that had been depleted of adenosine 5′-triphosphate by preincubation in glucose-free medium containing KCN and lodoacetate and thus did not phosphorylate the substrates. Transport of both nucleosides was so rapid that the intracellular concentration approached that in the extracellular fluid within less than 1 min. Initial transport velocities were computed from pseudo-first-order time courses of intracellular substrate accumulation as determined by a rapid mixing-sampling technique. The zero-trans Km was similar for both nucleosides, between 250 and 500 µm. The rates of transport of deoxycytidine and 1-β-d-arabinofuranosylcytosine into the cells were 10 to 100 times higher than their rates of intracellular phosphorylation in untreated cells in which phosphorylation was not prevented by adenosine 5′-triphosphate depletion. Thus phosphorylation rather than transport was the rate-determining step in their incorporation into the nucleotide pool in these cells. The intracellular phosphorylation of the nucleosides, however, became enhanced 5 to 10 times within minutes of addition of 0.1 mm thymidine or 0.5 to 1 mm hydroxyurea or of appropriate concentrations of other inhibitors of ribonucleotide reductase, imidazopyrazole, and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone to the medium. Pyrazofurin, an inhibitor of de novo pyrimidine synthesis, 3-deazauridine, an inhibitor of cytidine 5′-triphosphate synthestase, and alanosine, an inhibitor of the conversion of inosine 5′-phosphate to adenosine 5′-phosphate, had a similar but more delayed effect. The stimulation of deoxycytidine and 1-β-d-arabinofuranosylcytosine incorporation into the nucleotide pool resulted to varying extents in their enhanced incorporation into DNA depending on the degree of inhibition of DNA synthesis caused by the various treatments. The results indicate that the intracellular deoxycytidine 5′-triphosphate concentration is normally high enough to cause severe feedback inhibition of deoxycytidine kinase. A decrease in 5′-triphosphate concentration due to inhibition of its de novo synthesis caused by thymidine, 3-deazauridine, or pyrazofurin results in enhancement of the salvage pathway. The effect of ribonucleotide reductase inhibitors and of alanosine, on the other hand, may be related to the depletion of the cells of deoxyadenosine 5′-triphosphate.