Showing papers in "Cancer Research in 1982"

Induction of Microsomal Enzymes by Foreign Chemicals and Carcinogenesis by Polycyclic Aromatic Hydrocarbons: G. H. A. Clowes Memorial Lecture

Abstract: extremely grateful to the Burroughs Wellcome Co. and to Hoffmann-La Roche Inc. for their support of my research in Tuckahoe from 1960 to 1970 and in Nutley from 1970 to the present. Looking back at the names of earlier recipients of the Clowes Award, I realize that I am the 6th Clowes Lecturer who, at one time or another in his career, has been associated with the McArdle Laboratory for Cancer Research at the University of Wisconsin. I believe that the large number of awardees associated with McArdle is telling us something about the genius of Dr. Harold Rusch who established McArdle with a handful of outstanding young investigators. Dr. Rusch helped to develop in his associates an intense devotion to fundamental cancer research, and he provided his colleagues with an at mosphere that encouraged them to have critical but construc tive interactions with each other in ways that stimulated excel lence in research by all of the members of the group. In 1952, I had the good fortune of coming to McArdle as a graduate student of Drs. James and Elizabeth Miller. Although I failed in my first research assignment, which was to synthesize pure 2amino-1-naphthol, the Millers had great patience with me, and they tried very hard to instill their high standards into my research. Whatever small accomplishments I may have made to biomédical research were in large part the result of the excellent training and encouragement that I received from Jim and Betty Miller. An important problem that has been of great interest to me for many years is individuality in the response of human beings and other living organisms to foreign chemicals. Why does a drug or an environmental pollutant cause toxicity in one person and not in another person? Why do some cigarette smokers develop lung cancer whereas other cigarette smokers do not? One of the causes of variability in the response of human beings to a foreign chemical is individuality in the rate of metabolism of the chemical. By the mid-1960s, it was recog nized by clinical pharmacologists that the plasma half-lives and steady state plasma concentrations of drugs varied by as much as 10to 20-fold among different individuals (42, 115, 204, 318). Because it seemed likely that there were also large differences in the metabolism of chemical carcinogens in dif ferent individuals, we investigated the metabolism of BP2 by

Growth of human hepatoma cells lines with differentiated functions in chemically defined medium.

Abstract: A human hepatoma cell line, HuH-7, which was established from a hepatocellular carcinoma, was found to replicate continuously in a chemically defined medium when the medium was supplemented with Na2SeO3. The cells grew better in this medium than in serum-containing medium without any adaptation period. Other established human hepatoma and hepatoblastoma cell lines, HuH-6 cl-5, PLC/PRF/5, huH-1, and huH-4, also grew in the defined medium. Although HLEC-1 cells failed to proliferate continuously with Na2SeO3 alone, they grew if a cell-free conditioned medium from HuH-7 cells was added to the medium. These cell lines, except the HLEC-1 cell line, produced the following human plasma proteins among those examined: albumin, prealbumin, alpha 1-antitrypsin, ceruloplasmin, fibrinogen, fibronectin, haptoglobin, hemopexin, beta-lipoprotein, alpha 2-macroglobulin, beta 2-microglobulin, transferrin, lipoprotein, alpha 2-macroglobulin, beta 2-microglobulin, transferrin, Complement Components 3 and 4, and alpha 1-fetoprotein. Beside plasma proteins, the media from HuH-7, HuH-6 cl-5, PLC/PRF/5, and huH-1 contained anti-carcinoembryonic antigen-reactive proteins, and those from PLC/PRF/5, huH-1, and huH-4 medium contained hepatitis B surface antigen. These proteins were detected during periods of serial cultivation over 9 months under the above culture conditions. The hepatoma cell lines grown in the fully defined synthetic medium may provide a new approach for investigating the growth and metabolism of human hepatoma cells in vitro.

Induction of Maturation in Cultured Human Monocytic Leukemia Cells by a Phorbol Diester

Abstract: Suspension cultures of a human monocytic leukemia cell line, THP-1, were treated with 0.16 to 160 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). In an original cell line, THP-1-O, cultured again from -80 degrees cryopreservation, more than 80% of the cells adhered to the glass substrate with marked morphological change within 3 hr of TPA treatment. Adherent cells became flat and amoeboid in shape, and many microvilli and flaps of the cell surface disappeared. Well-developed Golgi apparatus, rough endoplasmic reticula, and a large amount of free ribosomes were seen in the cytoplasm. On the other hand, in THP-1-R cells cultured continuously without cryopreservation for 26 months, approximately 80% of the cells adhered to the substrate 48 hr after TPA treatment. Round and ovoid shapes were kept in THP-1-R cells treated with TPA. Surface Fc receptors for immunoglobulin G were present on more than 90% of THP-1-O and THP-1-R cells and were little affected by treatment with TPA. Sixty to 70% of the TPA-treated THP-1-O and THP-1-R cells were able to phagocytize yeasts and immunoglobulin G-coated sheep erythrocytes. Less than 20% of the untreated THP-1 cells were able to phagocytize yeasts and immunoglobulin G-coated sheep erythrocytes. In histochemical staining, alpha-naphthyl butyrate esterase was enhanced after treatment with TPA. Lysozyme activity in culture supernatants was not affected by TPA treatment. When exposed to latex beads and TPA, increased 14CO2 production from [1-14C]glucose in THP-1-O cells was observed. These results indicate that, after treatment with TPA, human monocytic leukemia cells may be converted into mature cells with functions of macrophages.

Increased accumulation of vincristine and adriamycin in drug-resistant P388 tumor cells following incubation with calcium antagonists and calmodulin inhibitors

Abstract: Some calcium antagonists and calmodulin inhibitors enhance the intracellular levels of vincristine and Adriamycin in vincristine- and Adriamycin-resistant P388 leukemia cells by inhibiting their outward transport. The high intracellular drug accumulation was directly related to the enhancement of the cytotoxicity of the antitumor agents, and the vincristine and Adriamycin resistance in these cells was circumvented.

Endogenous Hormones as a Major Factor in Human Cancer

Abstract: Hormone-related cancers account for almost 30% of all cancer cases in the United States. Data from animal experiments and from epidemiological and endocrinological studies in humans support the hypothesis that the individual hormones which control normal growth of target organs can also create the proper conditions for neoplastic transformation. The concept that hormones can cause, i.e., increase the incidence of, human cancer is most developed for the four hormone-related cancers which are numerically the most important, namely, breast, prostate, endometrium, and ovary. Even for these sites, large gaps remain in our knowledge of the responsible hormones and the conditions which create the optimal opportunity for carcinogenesis. Although scanty, the available epidemiological evidence also suggests a hormonal role in the pathogenesis of testis cancer, thyroid cancer, and osteosarcoma. We believe that the primary prevention of all these cancers will probably depend on modification of the factors which affect the secretion and metabolism of the responsible hormones rather than on control of exposure to classical exogenous initiators.

Copper- and Zinc-containing Superoxide Dismutase, Manganese-containing Superoxide Dismutase, Catalase, and Glutathione Peroxidase in Normal and Neoplastic Human Cell Lines and Normal Human Tissues

Abstract: Copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase form the primary enzymic defense against toxic oxygen reduction metabolites. Such metabolites have been implicated in the damage brought about by ionizing radiation, as well as in the effects of several cytostatic compounds. These enzymes were analyzed in 31 different human normal diploid and neoplastic cell lines and for comparison in 15 normal human tissues. The copper- and zinc-containing superoxide dismutase appeared to be slightly lower in malignant cell lines in general as compared to normal tissues. The content of manganese superoxide dismutase was more variable than the content of the copper- and zinc-containing enzyme. Contrary to what has been suggested before, this enzyme did not appear to be generally lower in malignant cells compared to normal cells. One cell line, of mesothelioma origin (P27), was extremely abundant in manganese-containing superoxide dismutase; the concentration was almost an order of magnitude larger than in the richest normal tissue. Catalase was very variable both among the normal tissues and among the malignant cells, whereas glutathione peroxidase was more evenly distributed. In neither case was a general difference between normal cells and tissues and malignant cells apparent. The myocardial damage brought about by doxorubicin has been linked to toxic oxygen metabolites; particularly, an effect on the glutathione system has been noted. The heart is one of the tissues which have a low concentration of enzymes which protect against hydroperoxides. However, the deviation from other tissues is probably not large enough to provide a full explanation for the high doxorubicin susceptibility. In the present survey, no obvious relationship between generally assumed resistance to ionizing radiation or to radical-producing drugs and cellular content of any of the enzymes could be demonstrated.

Synthesis and Degradation of Heat Shock Proteins during Development and Decay of Thermotolerance

Abstract: Morris hepatoma 7777 cells, heat conditioned at 43 degrees for 0.5 hr, become gradually thermoresistant during an incubation at 37 degrees as judged by their ability to form colonies following a second heat challenge. Pulse incorporation of [35S]methionine into proteins at various times after the conditioning treatment and subsequent fractionation of the proteins by polyacrylamide gel electrophoresis indicate that the gradual putative modifications occurring at the cellular level and leading to the thermotolerance state are accompanied by an elevated synthesis above the normal level of a small set of polypeptides with apparent molecular weights of 27,000, 65,000, 68,000, 70,000, 89,000, and 107,000. Both thermotolerance development and protein induction are completed after a 6- to 8-hr period. At the end of this period, thermotolerance is at its maximum level and heat shock protein synthesis is returned to normal. This acquired thermal resistance eventually disappears between 60 and 80 hr following conditioning treatment. In a parallel manner, the heat shock-induced proteins synthesized during the first 4 hr following the conditioning treatment are maintained in the cells at a high level for several hr but become undetectable by 82 hr. The results provide strong circumstantial evidence that heat shock proteins are involved in the acquisition, maintenance, and decay of thermotolerance.

Failure of RNA synthesis to recover after UV irradiation: an early defect in cells from individuals with Cockayne's syndrome and xeroderma pigmentosum

Abstract: Previous work has shown that in cells from the ultraviolet-sensitive genetic disorder, Cockayne's syndrome, DNA synthesis fails to recover after ultraviolet irradiation, despite the fact that these cells have no detectable defect in either excision or daughter-strand repair pathways We now show that Cockayne cells, as well as cells from a number of patients with xeroderma pigmentosum, are sensitive to the lethal effects of UV irradiation in stationary phase under conditions in which no DNA is synthesized after irradiation Furthermore, in normal and defective human fibroblasts, RNA synthesis is depressed after UV irradiation In normal (dividing) cells, RNA synthesis recovers very rapidly, but this recovery does not occur in Cockayne cells, and it is reduced or absent in xeroderma pigmentosum cells from different complementation groups Qualitatively, similar results are obtained with cells in stationary phase The recovery of RNA synthesis in the various defective cell strains is not correlated with the overall extent of excision repair, but there is some correlation between recovery of RNA synthesis and cell survival after ultraviolet irradiation These results implicate recovery of RNA synthesis as an important early response to ultraviolet irradiation

Gastrointestinal cancer-associated antigen in immunoperoxidase assay.

Abstract: A monosialoganglioside antigen of gastrointestinal adenocarcinomas defined by murine monoclonal antibody was demonstrated by immunoperoxidase (IP) assay in fixed paraffinembedded tumors in 59% of colonic adenocarcinomas, 86% of pancreatic adenocarcinomas, and 89% of all gastric adenocarcinomas. In all patients with detectable levels of antigen in circulation, the resected tumors also expressed the antigen in IP assay. Six of eight individuals with no detectable levels of antigen in their serum samples expressed the antigen in the tumor tissue. Removal of the sialic acid residue of the antigen abolished the IP reaction. The successful use of the IP assay on fixed tissue to demonstrate the specific sites of gastrointestinal cancer antigen localization in human tumors and normal tissues provides an important tool for the study of developing neoplasia.

Tamoxifen and Metabolites in MCF7 Cells: Correlation between Binding to Estrogen Receptor and Inhibition of Cell Growth

Abstract: The binding of [3H]tamoxifen ([3H]Tam), a nonsteroidal antiestrogen, and of 4-[3H]hydroxytamoxifen ([3H]OH-Tam), a metabolite accumulated in vivo in target cell nuclei, was characterized in soluble extracts of human breast cancer MCF7 cells growing in a medium depleted in estrogens. Saturation analysis indicated a much higher affinity for OH-Tam (Kd = 0.15 nM) than for Tam (Kd = 4.8 nM). The binding of [3H]Tam and [3H]estradiol was competitive and mutually exclusive, and the binding site concentration (0.16 to 0.47 pmol/mg total protein) was similar for both ligands, strongly suggesting that antiestrogens were binding to the estrogen receptor (ER) in these cells. The ability of Tam and of some of its metabolites or derivatives to prevent the MCF7 cell growth was found to be correlated with their affinity for ER as determined by direct interaction or by binding competition with [3]estradiol on the uterine and MCF7 cytosol ER. OH-Tam was the highest-affinity compound and was 100-fold more active than Tam. The inhibitions observed were actually due to Tam and OH-Tam, respectively, since we did not detect any significant metabolism of these two labeled compounds by the MCF7 cells. N-desmethyltamoxifen, the other Tam metabolite found in high concentration in human plasma, was as effective as Tam while cis-tamoxifen appeared less effective. Compound E, which has no lateral chain, was the only tested compound having a good affinity for ER and a poor efficiency in preventing cell growth. These results support the hypothesis that antiestrogens control the growth of breast cancer by acting directly on the ER located in cancer cells.

Role of Laminin in the Attachment and Metastasis of Murine Tumor Cells

Abstract: We have studied the attachment of two murine metastatic cell lines and of a transformed, nonmetastatic sarcoma cell line to type IV (basement membrane) collagen. The metastatic cells attached preferentially to type IV collagen, whereas the nonmetastatic cells attached best to type I collagen. Laminin increased both the rate and the number of metastatic cells attaching to type IV collagen, while fibronectin had no effect. Antibodies to laminin prevented the attachment of metastatic cells to type IV collagen, while antibodies to fibronectin prevented the attachment of the nonmetastatic cells. The number of pulmonary metastases which formed after i.v. injection of cells into C57BL mice was used to measure the metastatic propensity of these cell lines. A subpopulation of the metastatic cells selected for by their ability to attach to type IV collagen in the presence of laminin produced more metastases than did unattached cells or cells attached with fibronectin. In addition, incubation of metastatic cells with antibody to laminin prior to injection into mice markedly reduced the number of lung metastases. These data suggest that laminin promotes the attachment of metastatic tumor cells to basement membrane during the metastatic process.

Analysis of the Fate of Systemically Administered Liposomes and Implications for Their Use in Drug Delivery

Abstract: Functional and ultrastructural studies of liposomes injected i.v. into inbred C57BL/6N mice were performed to determine whether free liposomes can traverse capillaries. In the liver and spleen, organs with discontinuous (sinusoidal) capillaries, ultrastructural and cell fractionation studies revealed that small (300- to 800-A diameter), sonicated, unilamellar liposomes were more efficient in penetrating liver sinusoids to interact with hepatocytes than were large (0.5- to 10-µm) multilamellar liposomes. Ultrastructural studies of the behavior of liposomes in the continuous capillaries of the lungs revealed that circulating phagocytic cells engulf the liposomes in the capillaries. Transcapillary migration of free liposomes was not observed. We conclude that free liposomes are unable to extravasate to reach the alveoli for subsequent engulfment by alveolar macrophages. Instead, liposomes in the lung capillaries are engulfed by circulating blood phagocytes which subsequently migrate to the alveoli to become alveolar macrophages. Experiments on the recruitment of blood monocytes into the lungs subjected to whole- or partial-body X-radiation confirmed that transfer of i.v.-injected liposomes to the alveolar compartment was mediated by blood monocytes. The inability of liposomes to escape from continous capillaries and their rapid uptake by circulating and fixed phagocytic cells calls into question the feasibility of using liposomes to “target” drugs to cells in extravascular tissues.

N-Nitroso compounds and childhood brain tumors: a case-control study.

Abstract: We questioned mothers of 209 young brain tumor patients and mothers of 209 controls about experiences of possible etiological relevance which they had during pregnancy or which their children had while growing up. Long-suspected brain tumor risk factors such as head trauma and X-rays appeared to be factors for relatively few cases. Increased risk was associated with maternal contact with nitrosamine-containing substances such as burning incense (odds ratio, 3.3; p = 0.005), sidestream cigarette smoke (odds ratio, 1.5; p = 0.03), and face makeup (odds ratio, 1.6; p = 0.02); with maternal use of diuretics (odds ratio, 2.0; p = 0.03) and antihistamines (odds ratio, 3.4; p = 0.002); and with the level of maternal consumption of cured meats (p = 0.008). These drugs contain nitrosatable amines and amides, and the cured meats contain nitrites, chemicals which are precursors of N-nitroso compounds. We propose a hypothesis that brain tumors in these young people are related to in utero exposure to N-nitroso compounds and their precursors, the most potent nervous system carcinogens known in experimental animals.

Distribution of Myoepithelial Cells and Basement Membrane Proteins in the Normal Breast and in Benign and Malignant Breast Diseases

Abstract: An immunocytochemical method for fixed and paraffin-embedded human breast biopsies is reported for the detection of myoepithelial and epithelial cells using antibodies to myosin and keratin, respectively, and of basement membranes using antibodies to laminin and type IV collagen. Using these markers, myoepithelial cells can be clearly distinguished in the normal breast and in the benign breast diseases sclerosing adenosis, epitheliosis, and fibroadenoma. In sclerosing adenosis, myoepithelial cells form a major cellular component. A stromally derived spindle cell is identified which stains with myosin but not with keratin antibodies (myofibroblast). These cells are seen in one-fifth of the fibroadenomas. Although cells staining with myosin antibodies are seen in the infiltrating component of all 18 carcinomas examined, elongated cells staining with both myosin and keratin antibodies (myoepithelial-like) are seen in only one infiltrating carcinoma where they are interposed at the stromal-epithelial junction of the infiltrating tumor cells. In contrast to the situation in benign breast diseases, mature myoepithelial cells form a very minor component of the majority of infiltrating ductal carcinomas. Basement membrane proteins, laminin, and type IV collagen are present in normal breast, benign breast disease, and grade I infiltrating ductal carcinomas but are absent in carcinomas of grades II and III.

Liposomes as in vivo carriers of adriamycin: reduced cardiac uptake and preserved antitumor activity in mice.

Abstract: Neutral and negatively charged liposomes containing Adriamycin (ADM) were examined for the efficiency of drug entrapment, serum stability, in vivo tissue distribution, and tumor-inhibitory activity. Entrapment of ADM in negatively charged liposomes containing phosphatidylserine (PS) or cardiolipin (DPG) was 3- to 4-fold higher than in neutral phosphatidylcholine (PC) liposomes with or without cholesterol (Chol). In the presence of human serum, PC:Chol and PS:PC:Chol liposomes were more stable than DPG:PC:Chol liposomes, as measured by the degree of release of entrapped drug. Tissue distribution studies after i.v. injection of liposome-entrapped ADM into mice indicated that the levels of ADM were increased severalfold in the liver and the spleen. Concomitantly, the concentration of ADM in the heart was significantly diminished when the drug was delivered by PS:PC:Chol and PC:Chol liposomes but only slightly reduced when DPG:PC:Chol liposomes were used. In vitro studies with primary cell cultures of a murine lymphoma showed that the cytotoxic activity of ADM was fully preserved with the various ADM-containing liposomes used. Finally, the in vivo antitumor activity of liposome-entrapped ADM was tested on a metastatic murine lymphoma. Large multilamellar PS:PC:Chol:ADM and PC:Chol:ADM liposomes, given as a single i.v. injection, were as effective in prolonging survival as equivalent doses of free ADM. Repeated i.v. injections of small sonicated PS:PC:Chol:ADM liposomes resulted in a significantly improved survival compared to free ADM. These results indicate that the delivery of ADM by certain types of liposomes may offer an efficient means of restricting its cardiac uptake and possibly minimizing its cardiotoxicity, while preserving its antitumor therapeutic activity.

Liver Regeneration Studies with Rat Hepatocytes in Primary Culture

Abstract: Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr after hepatectomy). Approximately 80% of the hepatocytes enter the cell cycle, and most of these cells go through mitosis. The replicating hepatocytes remain positive for glucose-6-phosphatase and negative for gamma-glutamyl transpeptidase, and they accumulate fat, in analogy to regenerating liver. Most of the replicating hepatocytes enter into multiple consecutive rounds of DNA synthesis. Dose-response studies between control animal serum and hepatocyte labeling index indicate that in unoperated animals the serum contains substances stimulatory as well as inhibitory for hepatic growth, with the inhibitory effect prevailing at high concentrations. After partial hepatectomy, the inhibitory activity disappears whereas the hepatopoietin activity reaches almost 90% of maximal biological effectiveness at 25% serum concentration. Addition of hormones to the system shows that the hepatopoietin activity is not identical to epidermal growth factor, platelet-derived growth factor, thyroxine, glucagon, or hydrocortisone. Norepinephrine abolishes the difference between control and hepatectomized serum but does not restore hepatopoietin activity when added to heat-inactivated serum. The results show that this system of replicating hepatocytes can be used to investigate the trophic factors that control growth of normal and neoplastic hepatocytes.

In Vivo Kinetics of Thymidylate Synthetase Inhibition in 5-Fluorouracil-sensitive and -resistant Murine Colon Adenocarcinomas

Abstract: The predictive utility of several biochemical parameters of 5-fluorouracil (5-FUra) action was evaluated in four murine colonic adenocarcinomas: 5-FUra-sensitive Tumor 38 and 5-FUra-resistant Tumors 07/A, 51, and 06/A. Thymidylate synthetase (TS) was determined by a tritiated 5-fluoro-2′-deoxyuridylate (FdUMP)-binding assay. Bolus 5-FUra (80 mg/kg, i.p.) administration caused in all tumors a rapid decrease in free TS levels. Only Tumor 38, however, showed inhibition of TS to undetectable (

Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopreserved lymphocytes.

Abstract: Blood samples from closely monitored patients at the Veterans Administration Hospital in Houston, Texas, were collected, coded, and sent to Microbiological Associates over an 8-month period. Lymphocytes were isolated and cryopreserved at -190 degrees. Lymphocyte samples were simultaneously thawed, phytohemagglutinin activated, and analyzed for benz(a)anthracene-induced aryl hydrocarbon hydroxylase (AHH) levels, [3H]thymidine incorporation, and reduced nicotinamide adenine dinucleotide-dependent cytochrome b5 (cytochrome c) reductase activity. Determinations were made at both 96 and 120 hr in culture, and peak activities were compared among a total of 51 individuals who expressed such lesions as squamous cell carcinomas (22%), adenocarcinomas (14%), oat cell carcinomas (6%), chronic obstructive pulmonary disease (22%), and other nonmalignant diseases. Of the 14 highest AHH/cytochrome c activities observed, all were found in patients with primary lung cancer. Mean AHH/cytochrome c activities were 0.89 for lung cancer patients (a total of 21) and 0.47 for noncancer patients (a total of 30) (p less than 0.001). No relationship was observed between AHH/cytochrome c activity and age of patient, numbers of cigarettes smoked, family history of cancer, location or histological type of tumor, or level of phytohemagglutinin blastogenesis ([3H]thymidine cpm/cytochrome c). Whether the higher AHH levels are the cause or the result of the primary lung cancer remains to be determined.

Damaging Effects of Fourteen Chemotherapeutic Drugs on Mouse Testis Cells

Abstract: The harmful effects of 14 chemotherapeutic drugs on spermatogenesis in the mouse have been evaluated by studies of testicular cell killing and morphological and genetic damage produced. Male mice were given drugs as single injections at various doses up to the toxic levels. Prednisone and 6-mercaptopurine produced little or no cytotoxicity. All other drugs tested killed differentiated spermatogonia. Of these, methotrexate, cyclohexylchlorethylnitrosourea, cis-platinum, and mechlorethamine did not show significant stem cell killing. Bischlorethylnitrosourea, chlorambucil, 5-fluorouracil, mitomycin C, antinomycin D, and procarbazine showed some stem cell killing. Triethylenethiophosphoramide (thio-TEPA) was the only drug in this group which killed large numbers of stem cells. Only 5-fluorouracil and cis-platinum killed spermatocytes, and only cis-platinum killed spermatids. Several drugs induced chromosome breaks in treated spermatocytes. Thio-TEPA was effective in inducing chromosome translocations in treated spermatocytes and probably also in spermatocytes which originated from surviving treated stem cells. It had been our hypothesis that the cytotoxic effects of these drugs on mouse testicular stem cells would correlate with the duration of azoospermia observed in patients. This was shown not to be the case. Thus, the cytotoxic effects of single injections of single chemotherapeutic agents on the mouse testis did not appear to be predictive of which drugs will cause long-term azoospermia in humans.

Antigens of Human Pancreatic Adenocarcinoma Cells Defined by Murine Monoclonal Antibodies

Abstract: We have elicited and characterized the serological specificity of five murine monoclonal antibodies (DU-PAN-1, 2, 3, 4, and 5) to a human pancreatic tumor cell line, HPAF. The antibodies are not detecting HLA-associated antigens since all of the monoclonals failed to react with human lymphoid and myeloid cell lines and uncultured cells. All of the monoclonals except DU-PAN-5 reacted with four of five pancreatic tumor cell lines and two of two uncultured pancreatic tumors. An immunoperoxidase technique was used to determine the presence of the antigens detected by the monoclonal antibodies in frozen sections of tumor and adult and fetal normal tissues. DU-PAN-1 antigen was detected on pancreatic tumors, and a transitional cell carcinoma of the bladder, but was detected on no other adult or fetal normal tissues including pancreas. One of the antigens (DU-PAN-2) defined by the monoclonals was present on pancreatic ductal epithelial cells and showed a restricted distribution on tumor cells from some other carcinoma patients and on cells from certain fetal tissues. DU-PAN-3 antigen was present on adult and fetal pancreatic cells and certain tumor cells but could not be detected on cells of other fetal or adult normal tissues. DU-PAN-4 and DU-PAN-5 antigens have a more widespread distribution on normal or tumor cell types.

Synergistic interaction of two classes of transforming growth factors from murine sarcoma cells.

Abstract: Transforming growth factors (TGFs) isolated from murine sarcoma virus-transformed 3T3 cells have been separated by high-pressure liquid chromatography into two subsets. One subset, called TGFα, competes with epidermal growth factor (EGF) for receptor sites, whereas the other, called TGFβ, does not. TGBβ, purified by high-pressure liquid chromatography, will not induce formation of large colonies of cells in soft agar in the absence of TGFα or EGF. However, the combined action of either TGFα or EGF (which by themselves are relatively ineffective in promoting growth of cells in soft agar) together with TGFβ results in a potent synergistic effect, with formation of large colonies. Chemically modified analogs of EGF also potentiate TGFβ activity to the extent that they bind to the EGF receptor. It is suggested that TGFβ may be an important mediator of the known effects of both TGFα and EGF on neoplastic transformation.

Divergent responses in epidermal basal cells exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

Abstract: Mouse epidermal basal cells can be selectively cultivated in medium with 0.02 to 0.09 mm Ca 2+ and can be induced to differentiate by medium containing 1.2 mm Ca 2+ . Basal cell cultures were studied to determine if all cells in this population responded identically to the skin tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA). Studies on the induction of the enzyme epidermal transglutaminase by TPA demonstrated a 2- to 4-fold increase in activity within 12 hr of exposure. This activity increase paralleled morphological differentiation in approximately 50% of the basal cell population, and differentiating cells sloughed from the culture dish within 24 to 48 hr as transglutaminase activity returned to basal levels. The cells which remained were resistant to induced differentiation by 1.2 mm Ca 2+ medium, in that they failed to demonstrate increased transglutaminase activity or decreased thymidine incorporation, both characteristics of control basal cells induced to differentiate by 1.2 mm Ca 2+ . Cells remaining after a single exposure to TPA did not respond to a second exposure with an induction of transglutaminase if the interval between exposures was 4 days. TPA-pretreated cells did not undergo a transient decrease in thymidine incorporation (characteristic of control cells) when exposed to TPA a second time but instead were directly stimulated to proliferate by the phorbol ester, indicating that such cells were not refractory to the promoter. When the treatment-free interval after TPA was extended from 4 to 10 days, transglutaminase inducibility was restored in basal cells to either TPA or 1.2 mm Ca 2+ as inducers. These results indicate that heterogeneity exists within the epidermal cell population and that exposure to phorbol esters induces differentiation in some cells, while stimulating proliferation in others. Such heterogeneous responses would cause a selective redistribution of the epidermal cell population and could lead to clonal expansion of initiated cells.

Formation of the Cross-Link 1-[N3-Deoxycytidyl],2-[N1-deoxyguanosinyl]-ethane in DNA Treated with N,N′-Bis(2-chloroethyl)-N-nitrosourea

Abstract: The cross-linked dinucleoside, 1-[N3-deoxycytidyl],2-[N1-deoxyguanosinyl]ethane, has been isolated from DNA which has been exposed to N,N'-bis(2-chloroethyl)-N-nitrosourea. It is probable that this structure is responsible for the interstrand cross-linking observed previously by physical methods. The modification is unique in that it cross-links DNA through two base-pairing positions and probably arises through the transfer of a chloroethyl group to one of the bases followed by a second reaction of this group with the other strand of DNA. Initial attack could be at the N3 position of deoxycytidine, the N1 position of deoxyguanosine, or possibly the O6 position of deoxyguanosine. Attack at the O6 position of deoxyguanosine would require an internal cyclization with the N1 position of deoxyguanosine before secondary reaction with the N3 position of deoxycytidine but would explain resistance to N,N'-bis(2-chloroethyl)-N-nitrosourea in cells capable of removing substituents on the O6 position of guanine.

Application of Quantitative Stereology to the Evaluation of Enzymealtered Foci in Rat Liver

Abstract: The mathematical science of quantitative stereology has established relationships for the quantitation of elements in three-dimensional space from observations on two-dimensional planes. This report describes the utilization and importance of such mathematical relationships for the quantitative analysis of focal hepatic lesions in terms relative to the volume of the liver. Three examples are utilized to demonstrate the utility of such calculations in the three-dimensional quantitation of hepatic focal lesions. The first is that of a computer-simulated experiment based on defined hypothetical situations. The simulations demonstrate the applicability of the computations described in this report to the evaluation of two-dimensional data from typical animal experiments. The other two examples are taken from actual experiments and involve the transplantation of hepatic cell populations into the liver of suitably prepared hosts and the quantitation of altered foci produced by initiation with diethylnitrosamine-partial hepatectomy followed by promotion with phenobarbital. The quantitation of altered foci by means of a two-dimensional analysis (simple enumeration of focal intersections/area of tissue section) is proportional to the quantitation of foci per volume of liver provided that the mean diameter of the foci for each treatment is sufficiently uniform, as exemplified in the text by the transplantation experiment. When such mean diameters are unequal as in the diethylnitrosamine-phenobarbital experiment described herein, quantitation from three-dimensional analysis gives significantly different results as compared with enumeration of focal intersections on two-dimensional areas. These studies clearly demonstrate that the frequency and size of foci intersections viewed on two-dimensional tissue sections do not necessarily reflect the number or size of foci in the three-dimensional tissue. Only by quantitating the number and size of the foci in relation to the three-dimensional volume of the tissue can one determine the validity of the proportionality of data from two-dimensional measurements to the total number of foci per volume of tissue. Such a conclusion has important implications for quantitative studies on hepatocarcinogenesis as well as for the enumeration of premalignant lesions which occur during the natural history of carcinogenesis in any solid tissue.

Interactions of Rhodamine 123 with Living Cells Studied by Flow Cytometry

Abstract: The cationic fluorochrome rhodamine 123 (R123), reported to bind specifically to mitochondria of living cells, was presently investigated with respect to its uptake by a variety of cell types in various functional states and the subsequent effect of the dye on cell growth. The emission spectrum of R123 taken up by cells undergoes a 12-nm red shift, suggesting formation of a complex. Cells accumulate R123 rapidly; near maximum binding is reached after 5 to 10 min, regardless of the temperature (0-37 degrees) of incubation. There is a dose-dependent relationship between R123 concentration in the medium and the dye accumulation in the cell that covers the range of 0.1 to 10.0 and 0.1 to 5.0 microgram of R123 per ml under equilibrium and nonequilibrium conditions, respectively. Some leakage of the dye from cells occurs, following their transfer into dye-free medium. Despite the leakage, the intracellular dye can be detected after at least two cell divisions, thus indicating that: (a) the R123-labeled cells divide; (b) during division, labeled mitochondria are distributed into the daughter cells; and (c) R123 may be used as a cell tracer. Cell death often is accompanied by a transient increase in R123 fluorescence. Dead cells exhibit either uniform, strong fluorescence or show a patchy labeling pattern suggesting swollen mitochondria. With time (4 to 8 hr), dead cells lose ability to retain R123 and lyse. Uptake of R123 by living cells is increased during the transition from quiescence into the cycle, and a decrease is seen when Friend leukemia cells undergo erythroid differentiation; in all cases, changes in R123 uptake are correlated with changes in cellular RNA content. Simultaneous cell staining with R123 and ethidium or propidium provides a rapid assay of the viability of the cells and their metabolic state, i.e., as related to proliferation or motility. Pulse-labeling of cells with up to 10 microgram of R123 per ml has no significant effect on their immediate growth and cloning efficiency. In the continuous presence of R123, however, cells become specifically arrested in the G1A compartment, i.e., in early G1 phase. Detailed analysis of the cell cycle kinetics reveals that cell progression through all phases is slowed 4 hr after addition of R123. Cell exit from G1A, however, is affected as early as 2 hr following addition of R123, and with time the cells are unable to leave this compartment at all. Uncharged rhodamine dyes (rhodamine 110 and rhodamine B) do not accumulate in mitochondria and are without effect on the cell cycle. The cytostatic effect of R123 is discussed in light of the dye specificity for mitochondrial membranes and the disruption of cell energy metabolism, resulting in the inability of the cells to attain a critical content of essential components (i.e., ribosomal RNA), necessary for cell entrance into the prereplicative (G1B) compartment of G1 phase.

Phase I and Pharmacological Studies of Adriamycin Administered Intraperitoneally to Patients with Ovarian Cancer

Abstract: A Phase I study of Adriamycin administered ip was performed in 10 ovarian cancer patients who were refractory to systemic chemotherapy Adriamycin was infused in 2 liters of Inpersol via a semipermanent Tenckhoff dialysis catheter Adriamycin was administered for a 4-hr dwell every 2 weeks with concentrations ranging from 9 to 54 µm The dose-limiting toxicity of ip Adriamycin was a sterile peritonitis Severe abdominal pain with ascites and adhesions was observed at concentrations greater than 36 µm There were three objective responses, and two other patients had a marked reduction in ascites formation while on treatment The objective responses were in patients who had small volume ( Adriamycin concentrations were measured by high-pressure liquid chromotography A mean of 85% of the drug was absorbed over the 4-hr dwell time The concentrations attained ip have been demonstrated previously to be cytotoxic to human ovarian cancer cells from untreated patients or from patients who had relapsed after treatment with a non-Adriamycin combination Plasma levels peaked within the first hr after ip instillation Plasma levels were markedly lower than corresponding peritoneal concentrations The maximum pharmacological advantage (peak peritoneal concentration/peak plasma concentration) was 474, while the 4-hr peritoneal level was 166 times higher than the corresponding plasma level after an ip dose of 40 mg/2 liters (36 /µm) The peak plasma levels after a 60-mg/2 liters (54 µm) dose were 10 times lower than after a 60-mg iv dose The recommended starting dose for a Phase II trial is 27 to 36 µm (30 to 40 mg Adriamycin per 2 liters Inpersol) with a 4-hr dwell every 2 to 3 weeks for six cycles

Induction of Differentiation of the Human Histiocytic Lymphoma Cell Line U-937 by Retinoic Acid and Cyclic Adenosine 3′:5′-Monophosphate-inducing Agents

Abstract: The monoblast-like human histiocytic lymphoma cell line, U-937, is induced to differentiate into monocyte-like cells by incubation with 0.1 to 1.0 µM retinoic acid (RA). These induced cells are phagocytic, reduce nitroblue tetrazolium, and show an increased hexose monophosphate shunt activity, consistent with monocyte-like cells.Prostaglandin E, cholera toxin, or dibutyryl cyclic adenosine 3′:5′-monophosphate, all inactive alone, increased markedly the extent of RA-induced differentiation of U-937. These data suggest that the intracellular level of cyclic adenosine 3′:5′-monophosphate is of importance for expression of the RA-induced effect. Responsiveness to RA seems to be limited to those leukemic myeloid cells blocked at a relatively late stage of maturation, like the promyelocytic HL-60 and the monoblast-like U-937 cell lines.

Cathepsin B activity in B16 melanoma cells: a possible marker for metastatic potential.

Abstract: In solid sc tumors of a variant of the murine B16 melanoma with high metastatic potential (B16F 10 ), there was a 2- to 7-fold elevation of lysosomal cathepsin B activity when compared to the B16F 1 variant with low metastatic potential The highest activities (based on either protein or DNA) of cathepsin B were found in tumors of less than 1 g When B16F 1 and B16F 10 melanoma variants were grown in tissue culture, the metastatic differential in cathepsin B activity was lost as the cells were subcultured However, this differential in cathepsin B activity could be restored by reestablishing the cultured cells as sc tumors The activities of four other lysosomal enzymes (cathepsin D, β- N -acetylglucosaminidase, β-glucuronidase, and acid phosphatase) showed little evidence of a positive correlation with the metastatic potential of the B16 melanoma variants Eighty to 90% of cathepsin B activity has been localized to a fraction containing viable tumor cells which was isolated by centrifugal elutriation In contrast, only 50% of cathepsin D activity was in the viable tumor cell fraction, and from 30 to 70% of β- N -acetylglucosaminidase, β-glucuronidase, and acid phosphatase Elevated levels of cathepsin B in the high metastatic B16F 10 variant are consistent with the idea that cathepsin B may play a direct or a regulatory role in tumor metastasis

In Vitro Cellular Effects of Hematoporphyrin Derivative

Abstract: Several in vitro cell systems were exposed to hematoporphyrin derivative (HPD): established lines of rat kangaroo epi thelial kidney; normal mouse embryonic fibroblasts; and differ entiated neonatal rat myocardial cells. The uptake of HPD (25 to 100 jiig/ml) by individual cells occurred rapidly over a 2-hr period and leveled off by 24 hr. HPD was excreted from cells by 48 hr after exposure. However, a low level of HPD (above background) was maintained in cells for up to 4 days following cessation of exposure. Intracellular binding of HPD was to mitochondria as demonstrated by fluorescence microscopy. HPD was also shown to have a growth-inhibiting effect on rat kangaroo cells without added light. The growth effects on mouse cells were less marked.

Susceptibility of Fanconi's anemia lymphoblasts to DNA-cross-linking and alkylating agents.

Abstract: In order to develop the usefulness of Fanconi's anemia (FA) lymphoblast lines for biochemical and genetic studies, we have determined their sensitivity to a variety of DNA-damaging chemicals. We have adapted a growth inhibiton protocol in which the sensitivity of a cell line is characterized by the drug concentration yielding a 50% inhibiton of growth (EC50). The DNA-cross-linking agents, mitomycin C, nitrogen mustard, melphalan, 1,3-butadiene diepoxide, cis-diaminedichloroplatinum(II), and cyclophosphamide, were all more toxic to four FA cell lines than to five normal lines. Three lines, HSC 72 (FA), 99 (FA) and 230 (FA), had EC50s that were 10 to 20 times lower than that of controls while the fourth line, HSC 62 (FA), had an intermediate EC50. Three nitrosourea compounds were also more toxic to FA cells than to controls. However, 2 normal cell lines (HSC 92 and 93) had nitrosourea EC50s 4 to 7 times lower than the other nine controls and overlapped the sensitivity of the intermediate [HSC 62 (FA)] cell line. The same 2 normal cell lines were also more sensitive than 12 other controls, including FA heterozygotes, xeroderma pigmentosum, and ataxia telangiectasis, to the monofunctional alkylating agents, ethyl methane sulfonate, methyl methane sulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine. Heterogeneity was also found with FA lines. Two FA cell lines (HSC 72 and 230) had EC50s lower than all control lines while one FA line (HSC 99) had an EC50 similar to that of the resistant normal lines. FA and normal cells had nearly the same sensitivity to 4-nitroquinoline-1-oxide and bleomycin. These results demonstrate that FA lymphoblast lines are more sensitive than normal cell lines to all DNA-cross-linking agents examined. These cell lines should therefore be useful for the analysis of DNA cross-link repair and the biochemical defect in FA. We have also found an unexpected sensitivity of some FA and normal lines to monofunctional alkylating agents.