Showing papers in "Cancer Research in 1981"

Overcoming of Vincristine Resistance in P388 Leukemia in Vivo and in Vitro through Enhanced Cytotoxicity of Vincristine and Vinblastine by Verapamil

Abstract: A noncytotoxic dose of verapamil, a coronary vasodilator, enhances the cytotoxicity of vincristine (VCR) and vinblastine in P388 leukemia and its VCR-resistant subline, P388/VCR. When 2.2 to 6.6 microM verapamil was added along the VCR to the P388/VCR culture in vitro, VCR resistance was completely overcome. Verapamil in doses of 50 to 100 mg/kg administered daily for 10 days with VCR also enhances the chemotherapeutic effect of VCR in P388- and, especially, P388/VCR-bearing mice. When approximately 3 times the amount of VCR was given to a P388/VCR bearer as compared to a P388 bearer, VCR resistance was almost completely overcome in vivo with 50 to 100-mg/kg doses of verapamil. The amount of VCR incorporated into P388 cells was larger than that in P388/VCR cells. Verapamil (6.6 microM) enhanced the cellular level of VCR in P388 cells 2-fold and enhanced the level of VCR in P388/VCR cells 10-fold. The amount of VCR in P388/VCR cells reached the same level as that found in P388 cells. The overcoming of VCR resistance in vivo and in vitro could be explained by the effective accumulation of VCR by verapamil in P388/VCR cells mediated by the inhibition of a VCR efflux function of the cells, a mechanism which remains to be solved.

Tumorigenic Keratinocyte Lines Requiring Anchorage and Fibroblast Support Cultured from Human Squamous Cell Carcinomas

Abstract: We have established cell lines from six human squamous cell carcinomas (SCC) of the epidermis and tongue, using culture methods previously developed for clonal growth and serial cultivation of normal keratinocytes. The SCC lines all form rapidly growing, well-differentiated SCC's or progressively growing squamous cysts in nude mice. In contrast to normal keratinocytes, SCC cells form unstratified or very poorly stratifying colonies and do not require epidermal growth factor for sustained growth. The SCC lines vary in their requirement for a fibroblast feeder layer to support clonal growth, as normal keratinocytes possess. Only one line forms large, progressively growing colonies at high efficiency in semisolid medium; the other five lines exhibit only a small amount of abortive growth in semisolid medium, after which the cells appear to rapidly degenerate. These results demonstrate that SCC's often grow as established lines in culture, but they frequently possess in vitro growth requirements similar to those of normal keratinocytes. Consequently, neither semisolid medium nor standard surface culture media are appropriate for initiating primary SCC cultures or for selecting transformants out of carcinogen-treated keratinocyte populations, because they do not provide conditions permissive for the growth of many malignant keratinocytes.

Effect of Plasminogen Activator (Urokinase), Plasmin, and Thrombin on Glycoprotein and Collagenous Components of Basement Membrane

Abstract: Tumor cells traverse basement membranes (BM) during the stages of the metastatic process. Penetration of the BM may involve proteolysis by enzymes directly or indirectly associated with tumor cells. This study evaluated the role of the serine proteases urokinase (plasminogen activator), plasmin, and another regulatory protease, alpha-thrombin, in the degradation of the BM. Homogeneously pure enzyme preparations were incubated with isolated components of BM and with whole human amnion BM. The BM components consisted of acid-extracted type IV collagen, pepsin fragments of collagen type IV, laminin, and fibronectin. Collagen type V (alpha A alpha B) associated with the peri-BM zone was also studied. The purity of the enzymes was verified by gel electrophoresis and inhibitor studies. Digestion of the BM components was performed at 25 degrees using matched activity for the different enzymes. Urokinase failed to significantly degrade fibronectin or any of the other BM components. Under the same 25 degrees (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or alpha A alpha B (type V) collagade fibronectin or any of the other BM components. Under the same 25 degrees (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or alpha A alpha B (type V) collagade fibronectin or any of the other BM components. Under the same 25 degrees (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or alpha A alpha B (type V) collagen. alpha-Thrombn selectively degraded only the m.w. 400,000 chain of laminin, whereas plasmin degraded both the laminin chains. Digestion of laminin by the serine proteases was time and concentration dependent, as verified by a new degradation assay using [14C]laminin. A variety of normal and neoplastic cells were tested for the presence of laminin-degrading proteases. macrophages, polymorphonuclear leukocytes, and metastatic tumor cells contained a significant laminin-degarding activity. The activity was enhanced by the addition of plasminogen. Type V collagen was cleaved by thrombin and plasmin at 35 degrees but not at temperatures below 33 degrees. Following treatment of whole-amnion BM with any of these enzymes, electron microscopy demonstrated preservation of the lamina densa. Immunohistology studies indicated that laminin, but not type IV collagen, was removed from the whole BM by plasmin treatment. The results suggest that these BM components are poor substrates for plasminogen activators and that plasmin alone is not sufficient to completely degrade the whole BM...

Classification of Antineoplastic Agents by their Selective Toxicities toward Oxygenated and Hypoxic Tumor Cells

Abstract: The cytotoxicities of a number of antineoplastic agents to oxygenated and hypoxic EMT6 mouse mammary tumor cells in culture were examined. Based on the relative sensitivities of cells under aerobic and hypoxic conditions, drugs were placed into three categories. Drugs that were preferentially toxic to cells under oxygenated conditions were classified as type 1 agents; this group includes bleomycin, procarbazine, streptonigrin, actinomycin D, and vincristine. Type 2 agents were those preferentially toxic to cells under hypoxic conditions. These include mitomycin C and Adriamycin. On the basis of other published reports, the glucose analogs, 5-thio-D-glucose and 2-deoxy-D-glucose, and the radiosensitizers, misonidazole and metronidazole, can also be placed in this category. Several antineoplastic agents showed no major preferential toxicity to cells under the conditions of oxygenation or hypoxia used in these experiments and were placed in a third class. This group (type 3) includes 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, cis-diamminedichloroplatinum(II), 5-fluorouracil, and methotrexate. The success of many combination chemotherapy and combined modality treatments may be due to their ability to kill both the hypoxic and aerobic cell populations of solid tumors. Future chemotherapeutic regimens for the treatment of solid tumors should include agents and modalities directed toward the hypoxic cell population of the tumor, as well as toward the proliferating and nonproliferating tumor cell compartments; a therapeutic approach to the selection of antineoplastic agents for use in combination based upon physiological considerations of the architecture of solid tumors is presented.

Lysis of Fresh and Cultured Autologous Tumor by Human Lymphocytes Cultured in T-Cell Growth Factor

Abstract: Human lymphocytes derived from the peripheral blood of patients with a variety of cancers were grown in T-cell growth factor (TCGF) and tested in a 4-hr 51 Cr microcytotoxicity assay against fresh and cultured autologous tumor, autologous cultured skin fibroblasts, and autologous fresh peripheral blood lymphocytes. Lymphocytes grown in TCGF caused significant lysis of autologous cultured tumor and fibroblasts but caused little lysis of fresh autologous peripheral blood lymphocytes in all of seven patients tested. This lytic activity against autologous cultured cells was not dependent on the source of serum used in culturing the lymphoid cells or the targets. Lymphoid cells grown in TCGF also were capable of causing selective lysis of fresh autologous tumor cells that had never been in culture in five of nine patients. Lymphoid cells growing in lectin-free TCGF caused selective lysis of autologous tumor in five of seven patients. These observations demonstrate that peripheral lymphoid cells grown in TCGF can be lytic for autologous cultured and autologous fresh tumor compared to the lysis of fresh autologous peripheral blood lymphocytes. The fact that these auto-reactive cells, lytic for tumor, may be expanded to large numbers in TCGF suggests a possible role for these cells in studies of the control of the cytotoxic response of activated cells to tumor and possibly in the immunotherapy of tumors as well.

Transforming Growth Factor Production by Chemically Transformed Cells

Abstract: Evidence is presented indicating that the chemically transformed AKR-MCA and C3H/MCA-58 murine cell lines produce “transforming growth factor(s)” capable of inducing a transformed morphology and the ability to grow in soft agar in nontransformed, anchorage-dependent indicator cells. Serumfree medium conditioned by exposure to the chemically transformed cells was chromatographed on a Bio-Gel P-60 column after dialysis and lyophilization. Using the nontransformed mouse AKR-2B cells as the indicator cells, a peak of soft agar growth-stimulating activity was detected in the molecular weight range of 10,000 to 12,000. The soft agar growth-stimulating activity in pooled fractions from the AKR-MCA cells was shown to be trypsin and dithiothreitol sensitive and relatively heat stable; the activity was not destroyed by heating to 56° for 30 min or to 100° for 3 min. The pooled material also caused stimulation of growth in the soft agar of rat NRK cells and stimulation of DNA synthesis in the AKR-2B cells. The quantity required to give significant competition for binding to the epidermal growth factor receptor was about one order of magnitude greater than that required for stimulation of soft agar growth. Further separation of these polypeptide(s) by carboxymethylcellulose chromatography revealed three apparent peaks of soft agar growth-stimulating activity. Epidermal growth factor receptor-competing activity cochromatographed with the early minor soft agar growth-stimulating peak, whereas the two major peaks of soft agar growth-stimulating activity had no associated detectable competition for epidermal growth factor binding to its receptor. The data indicate that at least a major portion of the transforming growth factors produced by the chemically transformed cells is different from those described previously in murine sarcoma virus-transformed mouse cells and human tumor cells.

Heterogeneity of Malignant Cells from a Human Colonic Carcinoma

Abstract: Three subpopulations of malignant cells were isolated from a primary cell culture of a single human colonic carcinoma. The variant cells were established as cell lines designated HCT 116, HCT 116a, and HCT 116b, respectively. In vitro characterizations of the variant lines included growth in 0.5% agarose and growth on confluent layers of mouse fibroblasts. HCT 116a showed the highest colony formation in agarose and on confluent fibroblasts, while colony formation by HCT 116 was higher than that of HCT 116b in both of these systems. All of the variant lines were tumorigenic in athymic nude mice given injections of 10 x 10(6) cells, but the time between inoculation and tumor development (latency period) was approximately 10 times longer for HCT 116b as for HCT 116a and 8 times longer than for HCT 116. HCT 116b was not tumorigenic at an inoculum of 5 x 10(6) cells, while both HCT 116 and 116a were tumorgenic at this level. However, HCT 116a was clearly more tumorigenic than was HCT 116 on the basis of the number of animals developing tumors at inoculate of both 10 x 10(6) and 5 x 10(6) cells and on the basis of their differences in latency periods. While all the cell lines had near diploid numbers of chromosomes, each line showed a distinct histological pattern when grown as xenografts in athymic nude mice.

Fluorometric method for rapid detection of DNA strand breaks in human white blood cells produced by low doses of radiation.

Abstract: DNA strand breaks can be detected with great sensitivity by exposing crude cell lysates to alkaline solutions and monitoring the rate of strand unwinding. As little as one strand break per chromosome can be detected. Previous methods for measuring strand unwinding have required physical separation of single- from double-stranded molecules. We now describe conditions under which unwinding can be monitored directly using a fluorescent dye, thus greatly simplifying the analysis. Breaks due to irradiation of blood samples by 60Co gamma-rays at doses as low as 0.05 to 0.1 gray (5 to 10 rads) were detectable. Rapid rejoining of strand breaks during in vitro incubation at 37 degrees could readily be observed following a dose of one gray. Since the procedure is very rapid and cells can be analyzed directly without the requirement for culturing or radiolabeling, the procedure could be useful in cancer chemotherapy if in vivo damage is to be monitored or for testing the in vitro sensitivity of cells to drugs.

Clinical Spectrum of Lymphoproliferative Disorders in Renal Transplant Recipients and Evidence for the Role of Epstein-Barr Virus

Abstract: Six renal transplant recipients with abnormal lymphoproliferative disorders were studied in an attempt to define their clinical features and the role of Epstein-Barr virus (EBV) in their pathogenesis. Patients were either teenage (three) or in the sixth decade (three). The younger patients presented an average of 3 months after transplantation with fever, sore throat, and lymphadenopathy; had been markedly immunosuppressed; frequently had preceding or concomitant cytomegalovirus infections; and two of three had a rapidly fatal course. The older patients presented an average of 5 years after transplantation while on maintenance immunosuppressive drugs; in two of three cases with an oropharyngeal tumor; and had a more indolent, but frequently fatal, clinical course. The most frequent sites of biopsy-proven involvement in these patients were lymph nodes (three), the oropharynx (three), liver (three), bone marrow (three), transplanted kidney (three), colon (two), and central nervous system (two). EBV-specific antibody titers including anti-viral capsid antigen IgG, anti-viral capsid antigen IgM, anti-early antigen, and anti-Epstein-Barr nuclear antigen were serially measured in all patients. Four patients demonstrated serological evidence of a primary (one) or reactivation (three) EBV infection. No patient had significant changes in anti-early antigen or anti-Epstein-Barr nuclear antigen titers. All three patients tested for oropharyngeal shedding of EBV were positive. A touch imprint of one tumor was stained for the presence of Epstein-Barr nuclear antigen, and a majority of cells were positive. EBV complementary RNA/DNA filter hybridization and/or viral DNA/DNA reassociation analysis performed on tumor biopsy specimens in five patients demonstrated multiple EBV genome equivalents per cell in all eight specimens tested. Clinical, pathological, serological, and molecular hybridization studies provide substantial evidence that EBV was the cause of these lymphoproliferative disorders occurring after renal transplantation. Impaired host defenses allow the EBV-transformed B-lymphocytes to escape normal control mechanisms. This impairment is variable and influenced by many factors resulting in the observed spectrum of disease. Cytogenetic changes, however, may also be important.

Polymorphic diffuse B-cell hyperplasias and lymphomas in renal transplant recipients.

Abstract: The lymphoproliferative processes that developed in five renal transplant recipients were studied in an attempt to characterize and classify them morphologically. Nine surgical specimens, hematological material on all patients, and autopsy specimens from three patients were available. Studies performed included: conventional histopathology; evaluation of cell markers (immunoglobulins and sheep erythrocyte, complement, and Fc receptors) and cytoplasmic immunoglobulins (peroxidase-antiperoxidase technique); ultrastructural examination; and karyotype analysis. The lymphoid lesions in our patients shared marked cytological polymorphism (small and large cells, of both follicular center and “medullary” type) and polyclonal B-cell features, which indicated a common reactive nonneoplastic origin. However, other features, such as morphological atypia of the immunoblasts, extensive necrosis, chromosomal aberrations, and an incipient monoclonal component suggested the development of lymphoma in some of these lesions. In contradistinction, the abundance of typical immunoblasts was a feature that seemed to correlate with the clinical activity of the disease rather than with the biological malignancy. The multiplicity of B-cell types and the presence of a follicular center cell component with diffuse distribution, as well as the extensive necrosis in the malignant forms, seem to differentiate morphologically the lymphoproliferative processes arising in transplant recipients from both the hyperplasias and the lymphomas developing in immunologically normal hosts. For the former, we propose the terms of “polymorphic diffuse B-cell hyperplasias” and “polymorphic B-cell lymphomas.”

Cancer immunology: the search for specificity--G. H. A. Clowes Memorial lecture.

Abstract: The major focus of cancer immunology has shifted away from arguments about the validity of the immunosurveillance theory of cancer to the more basic question of tumor-specific antigens. Despite vast effort aimed at demonstrating such antigens, their existence in the generality of cancer remains unproven. Serological analysis of three tumor types, mouse leukemia, mouse sarcoma, and human malignant melanoma, has received most attention, and a rudimentary classification of the surface antigens expressed by these tumors has begun to emerge. The prime candidates for antigens that can be considered tumor specific are the few instances of Class 1 antigens that have now been serologically defined on mouse and human tumors. These antigens show an absolute restriction to individual tumors, not being demonstrable on any other normal or malignant cell type. Biochemical and genetic characterization of Class 1 antigens represents an essential next step in evaluating the significance of these antigens. The surprising features of the Thymus Leukemia (TL) antigens of the mouse provide insight into the genetic origin of another key class of tumor antigens, in this case antigens with characteristic properties of both differentiation antigens and tumor-specific antigens. In normal mice, TL antigens are restricted to cells in the thymus, and strains differ with regard to expression versus nonexpression of TL antigens. Genetic information for TL is universal in the mouse, however, as leukemias developing in mice that normally lack TL are found to express TL. What is clear from the past two decades of research in cancer immunology is that a far more detailed knowledge of surface antigens of tumor cells will be necessary before we can begin to assess the possibility of immunological control of cancer.

Adaptation versus Selection as the Mechanism Responsible for the Relapse of Prostatic Cancer to Androgen Ablation Therapy as Studied in the Dunning R-3327-H Adenocarcinoma

Abstract: The Dunning R-3327-H rat prostatic adenocarcinoma is a well-differentiated, slow-growing, serially transplantable tumor of spontaneous origin. When intact male rats bearing such an exponentially growing H-tumor s.c. are castrated, tumor growth abruptly stops, demonstrating the initial androgen sensitivity of this tumor. Eventually, however, after an extended period, the tumor invariably relapses and once again appears to grow exponentially. At the time of relapse, the tumor is no longer androgen sensitive but has irreversibly progressed to a completely insensitive state. The mechanism responsible for this irreversible progression has been demonstrated by fluctuation analysis not to be due to environmentally induced adaptation of initially androgen-dependent H-tumor cells to a new androgen-independent state. Instead, the progression is due to the basic heterogeneity of the original H-tumor (i.e., it is composed of a mixture of preexisting clones of both androgen-dependent and androgen-independent tumor cells). Following castration, only the preexisting clones of androgen-independent tumor cells are able to continue exponential growth; the androgen-dependent tumor cells stop proliferating and die. Thus, androgen ablation creates a host environment in which the androgen-independent tumor cells have a highly selective growth advantage over the androgen-dependent cells. Eventually, with time, this selective growth advantage results in a tumor which is completely composed of androgen-independent cells. It is the continuous proliferative growth of these androgen-independent tumor cells which leads to the relapse phenomenon.

Cytogenetic features of human neuroblastomas and cell lines.

Abstract: We reviewed the banded karyotypes of 24 human neuroblastomas and cell lines to identify any consistent chromosomal abnormalities. Six of the 10 primary tumors and one of the 14 cell lines were studied at this institution. Of the 24 neuroblastomas karyotyped, 20 were near-diploid, one was near-triploid, and 3 were near-tetraploid. One primary tumor had a diploid karyotype without numerical or structural rearrangements. The 20 cases with a karyotype in the diploid range were statistically analyzed for gain or loss of while chromosomes and for structural abnormalities of each chromosome arm. The short arm of chromosome 1 was preferentially involved in structural rearrangements, occurring in 14 cases (p less than 0.01). In 11 of these cases, the abnormality of chromosome 1 included deletion of bands 1p32 leads to 1pter, rendering the cells monosomic for this genetic material. Of the remaining three cases, one involved a reciprocal translocation of chromosomes 1p and 12q, another had insertion of genetic material at band 1p13, and the third had an extra dark band at 1p36. No other numerical or structural abnormalities occurred with sufficient frequency to reach statistical significance (p greater than 0.20). Six of the primary tumors or cell lines in the diploid range had double minute chromatin bodies, four cell lines had homogeneously staining regions, and two cell lines had either double minute chromatin bodies or homogeneously staining regions in subpopulations of cells. Hence, partial monosomy for the short arm of chromosome 1 was the most consistent cytogenetic abnormality in the human neuroblastomas studied.

Quantitative estimation of endogenous nitrosation in humans by monitoring N-nitrosoproline excreted in the urine.

Abstract: Endogenous formation of N-nitrosoproline (NPRO) was demonstrated by monitoring its excretion in the urine of a male volunteer who had ingested vegetable juice, as a source of nitrate, and proline. The resulting NPRO was analyzed after derivatization by combined gas-liquid chromatography thermal energy analysis. The amount of total NPRO excreted in the urine was found to be proportional to the proline dose and increased exponentially with the nitrate dose ingested. Neither nitrate nor proline, when taken alone, led to a detectable increase in NPRO in urine. The amounts of NPRO formed (as estimated from the amounts excreted within 24 hr) after dosing 325 mg nitrate (NO3-) followed by 500 mg proline, ranged from 16.6 to 30.0 (mean, 23.3) micrograms per person. The simultaneous intake of ascorbic acid or alpha-tocopherol inhibited nitrosation of proline in vivo. Monitoring of NPRO or other N-nitroso compounds excreted in the urine thus appears to be a suitable procedure for estimating daily human exposure to endogenously formed N-nitroso compounds.

Heterogeneous Oxygen Partial Pressure and pH Distribution in C3H Mouse Mammary Adenocarcinoma

Abstract: Severe disturbances in microcirculation during advanced phases of tumor growth lead to restrictions of convective and diffusive transport. In addition, an inhomogeneous distribution of transport conditions develops, resulting in insufficient and heterogeneous substrate supply and an inadequate drainage of wastes. Polarographic measurements of the local tissue oxygen tension (PO 2 ) using gold microelectrodes reveal that very low PO 2 values are prevalent in C3H mouse mammary carcinomas. The tissue PO 2 frequency distributions are shifted to low PO 2 values and limited in variability. The mean PO 2 value is 7 mm Hg. The median is 4 mm Hg, the modal class being 0 to 5 mm Hg. Within different microareas of the same tumor, pronounced heterogeneities exist. Due to an elevated rate of lactic acid production and its subsequent inadequate removal, a severe tissue acidosis is evidenced in malignant tumors. For C3H mouse mammary carcinomas, most of the measured pH values are in the range of 6.4 to 7.1, the modal class being 6.7 to 6.8 (mean pH, 6.73; median pH, 6.75). Within different microareas of the same tumor, clear heterogeneities in the pH distribution do occur. Very low pH values (5.8 to 6.3) have been observed in large ulcerated tumors. In extensively necrotic areas, pH values even higher than the arterial pH could be detected.

Testicular Cancer as a Model for a Curable Neoplasm: The Richard and Hinda Rosenthal Foundation Award Lecture

Abstract: The combination of platinum, vinblastine, and bleomycin was first used at Indiana University in 1974. Thirty of 47 patients (64%) survived for 5 years, and 27 (57%) are currently disease free (NED) and cured of their neoplasm. From 1976 to 1978, 78 consecutive patients were entered on a random prospective study that indicated that equal therapeutic results could be achieved with a lower dosage (0.3 mg/kg) of vinblastine. Fifty-two (67%) patients are continuously NED, and 57 (73%) are currently NED for 2 or more years. Our third-generation study, done in conjunction with the Southeastern Cancer Study Group, tested the hypothesis of whether maintenance vinblastine was necessary to ensure optimal cure rates in disseminated testicular cancer. One hundred thirteen patients entered this maintenance study, and the results demonstrated that cure in a far-advanced cancer could be achieved with only 12 weeks of therapy (remission induction) because the relapse rate in such patients was only 7%. The cure rate for patients presenting with locoregional disease (Stages A and B) should approach 100%. Platinum, vinblastine, and bleomycin will regularly produce a 70% complete remission rate, and a further 10% of patients will be rendered NED with surgical resection of residual disease. The relapse rate with four courses of remission induction therapy in a large cooperative group study (Southeastern Cancer Study Group) was only 7%. The high success rate in disseminated disease has allowed the option of high cure rate in Stage B disease (positive retroperitoneal nodes) with or without adjuvant chemotherapy. At Indiana University, 137 patients have been followed with Stage A and B nonseminomatous testicular cancer from 1973 to 1979 with a minimum follow-up of 2 years, and currently 135 are alive and well. Successful treatment strategies in testicular cancer have yielded a cure rate unparalleled in cancer treatment.

Autoradiographic distribution of hematoporphyrin derivative in normal and tumor tissue of the mouse.

Abstract: The distribution of isotopically labeled hematoporphyrin derivative (HPD) has been studied in mice bearing the spontaneous mammary tumor (fast growing). In stomach, liver, spleen, and pancreas, 3 hr after i.p. injection of [3H]HPD, grains were uniformly distributed over the tissue sections. After 24 hr, the grain density overlying parenchymous areas of these tissues was lower than that over the stromal or reticuloendothelial areas. In the spontaneous mammary tumor (fast growing), higher grain densities were seen over pseudocapsule, stromal septa, and necrotic areas at 3, 6, 12, 24, and 48 hr after injection. At 168 hr postinjection, only isolated stromal cells, presumably macrophages, showed high grain densities. From the temporal changes observed in the distributions of HPD in normal tissues and the relative stability of the distribution seen in the spontaneous mammary tumor (fast growing), we speculate that tissue factors such as vascular permeability, lack of an adequate lymphatic drainage, and nonspecific binding of serum proteins to stromal elements may be responsible for or contribute to the preferential uptake and/or retention of HPD observed in both human and animal tumors.

Melatonin inhibition and pinealectomy enhancement of 7,12-dimethylbenz(a)anthracene-induced mammary tumors in the rat.

Abstract: The effects of the pineal hormone, melatonin, and of pinealectomy on the incidence of mammary adenocarcinoma in Sprague-Dawley rats treated with 7,12-dimethylbenz(alpha)-anthracene (DMBA) were investigated. Melatonin (2.5 mg/kg), begun on the same day as DMBA (5 mg) treatment and given daily in the afternoon for 90 days, significantly reduced the incidence of mammary tumors from 79% (control) to 20% (treated) (p less than 0.002). Rats pinealectomized at 20 days of age and treated with 7 mg of DMBA at 50 days of age had a higher incidence of tumors (88%) compared to control animals (22%). Fifteen mg of DMBA, which resulted in a higher incidence of tumors, reduced the difference between pinealectomized and control animals. Melatonin only partially reversed the effects of pinealectomy, reducing the incidence from 87% (pinealectomy alone) to 63% (pinealectomy plus melatonin); however, the tumor incidence was still lower (27%) in nonpinealectomized, melatonin-treated animals. Assessment of plasma prolactin, luteinizing hormone, follicle-stimulating hormone, estradiol, and cortisol in DMBA-treated tumor-free and tumor-bearing animals revealed a significantly lower plasma prolactin concentration [27 +/- 5 (S.E.) ng/ml] in melatonin-treated animals as compared to vehicle-treated animals [65 +/- 8 ng/ml]. The concentration of plasma prolactin was less in melatonin-treated, pinealectomized rats (55 +/- 10 ng/ml) as compared to vehicle-treated, pinealectomized animals (101 +/- 13 ng/ml). Other hormones were not affected by melatonin treatment. These data support the hypothesis that melatonin inhibits the development of DMBA-induced mammary tumors in the rat while removal of the pineal gland stimulates development of such tumors. Additionally, these experiments provide evidence that these effects may be mediated by a suppression of plasma prolactin levels.

Isolation, Karyotype, and Clonal Growth of Heterogeneous Subpopulations of Human Malignant Gliomas

Abstract: The histological pleomorphism of human malignant gliomas as well as their variable response to therapy suggests that such tumors are not homogeneous but are composed of a heterogeneous population of cells. To search for heterogeneity in these tumors, we developed a protocol to identify by karyotype, isolate, and clone the subpopulations of the tumor. Freshly resected tumors were mechanically dissociated into single cells that were grown in suspension and short-term monolayer cultures. Chromosomal preparations were collected over the first 6 to 72 hr postresection. The karyotypes prepared comprised the “reference set” of chromosomes found in each tumor. The dissociated single cells were also dilution plated for monolayer culture. Cells that attached were marked and isolated as clones whose karyotypes were compared to those in the reference set in order to identify those clones that were cellular representatives of the tumor. Explant cultures established from the same tumor were grown in monolayer culture and cloned; their karyotypes were likewise compared to those in the reference set. Eight gliomas were analyzed. Each had metaphases that ranged in chromosome number from hypodiploid to hyperploid, but the frequency distribution of the subpopulations differed among the tumors. Defining a cellular subpopulation in the tumor as one yielding at least five karyotypically identical cells in the reference set, we found that each tumor contained from three to 21 subpopulations. All but one tumor produced 100 or more clones, and in seven tumors, more than 50% of the clones could be expanded for karyotyping; 7.6 to 25% of these clones had karyotypes identifiable in the reference set of their tumor. There was little relationship between the morphology of the clones and their karyotypes or growth kinetics. While explant cultures grew well, no clone derived from such cultures could be identified in the reference set. The protocol permits studies of clonal lines identifiable as cellular representatives of a patient9s tumor and not variants generated in tissue culture. These studies confirmed that human malignant gliomas are comprised of karyotypically heterogeneous cellular subpopulations. Preliminary work suggests that their phenotypic behavior may be similarly heterogeneous.

Effect of excess folates and deoxyinosine on the activity and site of action of 5-fluorouracil.

Abstract: The present study concerns the basis of the effects of high levels of folinic acid (CF) and deoxyinosine (dIno) on the potency and site of action of 5-fluorouracil (FUra) in mouse sarcoma (Sarcoma 180) and human carcinoma cells (Hep-2) in vitro. Sarcoma 180 cells are 50 times more sensitive to FUra than are Hep-2 cells, and thymidylate synthetase (dTMP synthetase) inhibition is growth limiting in Sarcoma 180 but not in Hep-2. Cells were exposed for 3 hr to varied concentrations of FUra alone or in combination with 10 µm CF and/or 1 mm dIno, and the following parameters were determined: (a) the subsequent growth (5 or 6 days) in FUra-free Medium 1640 with or without either 30 µm thymidine or 1 mm 2′-deoxyuridine (dUrd); (b) metabolism of FUra; (c) activity of dTMP synthetase in cell extracts; and (d) incorporation of [2-14C]dUrd at 0, 3, 6, 24, and 48 hr following FUra exposure. In both cell lines, dIno increased the cellular content of unbound 5-fluoro-2′-deoxyuridine-5′-monophosphate up to 100 µm and the inhibition of dTMP synthetase. The potency of FUra, as measured by growth inhibition, was increased only against Sarcoma 180 and not against Hep-2. A very rapid recovery of enzyme activity occurred after dIno, and dTMP synthetase inhibition did not become growth limiting for Hep-2. In both cell lines, 10 µm CF (1000 times more than required for growth) potentiated 3-fold the growth inhibition by FUra. This excess CF did not affect the extent of dTMP synthetase inhibition when measured immediately after FUra. Instead, the potentiating effect of CF is due to the stabilization of the enzyme-5-fluoro-2′-deoxyuridine-5′-monophosphate complex as evidenced by (a) a marked decrease in the ability of 1 mm dUrd to rescue Sarcoma 180 cells after treatment with FUra or Hep-2 cells after treatment with 5-fluoro-2′-deoxyuridine, without effect on the rescue with 2′-deoxythymidine and (b) a marked slow-down in the spontaneous recovery of dTMP synthetase activity after FUra or after FUra and dIno. In all conditions, the recovery of dTMP synthetase activity reached a limit at 6 hr after FUra removal. An impressive correlation was observed in Sarcoma 180 cells between the dTMP synthetase activities (measured by dUrd incorporation), which were reached 6 hr after FUra removal, and growth, which was measured at 5 days. Effects identical to those of 10 µm CF were obtained with 300 µm folic acid or 10 µmN5-methyltetrahydrofolic acid. A maximum potentiation of growth inhibition (10-fold) occurred after combinations of FUra with excess folates and dIno. It appears that the rate of recovery of dTMP synthetase activity plays a more significant role in the potency and site of action of FUra than does the amount of 5-fluoro-2′-deoxyuridine-5′-monophosphate which is formed and that this recovery can be greatly retarded with an excess of vitamins such as folate or CF.

Activation and Inhibition of Benzo(a)pyrene and Aflatoxin B1 Metabolism in Human Liver Microsomes by Naturally Occurring Flavonoids

Abstract: The effects of several naturally occurring and synthetic flavonoids on the metabolism of benzo(a)pyrene and aflatoxin B1 were evaluated. Addition of apigenin, chrysin, fisetin, flavonone, galangin, hesperitin, kaempferol, morin, myricetin, haringenin, or quercetin to human liver microsomes inhibited the hydroxylation of benzo(a)pyrene. In contrast to these results, the addition of flavone, nobiletin, tangeretin, or 7,8-benzoflavone to human liver microsomes caused a many-fold stimulation in the hydroxylation of benzo(a)pyrene, the metabolism of aflatoxin B1 to 2,3-dihydro-2,3-dihydroxyaflatoxin B1, and the metabolic activation of aflatoxin B1 to mutagenic products. Quercetin, morin, and kaempferol inhibited cytochrome c (P-450) reductase in human liver microsomes whereas flavone and 7,8-benzoflavone had no effect. These results suggest that the inhibitory effects of quercetin, morin, and kaempferol on monooxygenase activity may be caused at least in part by an inhibition in the reduction of cytochrome P-450. An examination of the structural features required for the inhibition and stimulation of benzo(a)pyrene hydroxylation indicated that all of the 12 flavonoid inhibitors that were studied possessed hydroxyl groups whereas the flavonoid activators were less polar molecules that lacked hydroxyl groups.

Basement Membrane Changes in Breast Cancer Detected by Immunohistochemical Staining for Laminin

Abstract: The distribution of the basement membrane glycoprotein laminin was studied by the immunoperoxidase technique in benign and malignant human breast tissue and in axillary lymph nodes from patients with breast cancer. An antiserum prepared against rat laminin was used. The specificity of this antiserum against human laminin was studied using the FL cell line of human epithelial-like cells derived from normal amniotic membrane. The antiserum reacted with these cells in immunoperoxidase staining and precipitated metabolically labeled secreted polypeptides which comigrated with polypeptides with molecular weights of 400,000 and 200,000 of rat laminin in sodium dodecyl sulfate:polyacrylamide gel electrophoresis. The neoplastic cells in malignant breast tissues showed strong cytoplasmic staining for laminin, and a positive reaction was aslo found in lymph node metastases. In some cases in which only micrometastases were present, these cells also stained strongly for laminin. In nonmalignant breast tissues, the epithelial cells of the duct were positive for laminin, but the staining was weaker than in the carcinomas. Pretreatment of the fixed tissue sections with trypsin markedly enhanced the staining of basement membranes for laminin. In trypsin-treated sections of normal breast tissue and benign lesions, the laminin staining delineated continuous basement membranes. In carcinomas representing the more differentiated types, basement membranes presumably produced by the tumor cells could be revealed by laminin staining, but they were thinner and discontinuous. The poorly differentiated carcinomas lacked organized basement membranes detectable by laminin staining. Our studies suggest that staining for laminin may be a useful adjunct test for detection of micrometatases in lymph nodes. The correlation of disintegration of the laminin-containing basement membranes of tumors with increasingly anaplastic appearance supports the notion that basement membranes may play a role in tumor invasion.

Inhibition of development of methylnitrosourea-induced rat colon tumors by indomethacin treatment.

Abstract: This study deals with the nonsteroid antiinflammatory drug indomethacin, a prostaglandin synthesis inhibitor, in the treatment of carcinogen-induced colonic tumors in rats. CD-Fischer rats were given an intrarectal instillation of methylnitrosourea (2 mg, three times a week from the first to the fifth week) in order to produce colonic tumors. Thereafter, a long-term treatment with an i.p. injection of indomethacin (2.5 mg/kg body weight, three times a week) was started at the 11th week, before the tumors developed. At autopsy, after the 15-week treatment with indomethacin, the colonic tumor incidence was significantly lower in treated rats than in vehicle-treated or untreated control rats. However, the 10-week cessation of treatment following this effective dosage led to a rapid development of tumors. In another experimental group of rats in which tumors were detected endoscopically, when treatment was initiated late, at the 26th week, for 10 weeks, tumor incidence and number were not altered. The data suggested that indomethacin inhibited the development of colonic tumors but did not reject or kill the growing tumors. It is postulated that indomethacin treatment may effectively prevent the development of colonic cancer in patients at high risk of colonic adenomatosis and carcinomatosis.

DNA Cross-Linking as an Indicator of Sensitivity and Resistance of Mouse L1210 Leukemia to cis-Diamminedichloroplatinum(II) and l-Phenylalanine Mustard

Abstract: The relationship between DNA cross-linking and cell killing by cis -diamminedichloroplatinum(II) ( cis -DDP) and l-phenylalanine mustard (L-PAM) was studied in L1210 cell culture lines and in mice bearing sensitive and resistant lines of L1210 leukemia. A line of L1210 mouse leukemia cells was developed which is resistant to cis -DDP in vitro . These cells, designated ZCR9, are cross-resistant to L-PAM. The effect of both drugs on the ZCR9 cells, compared to the parent L1210 K25 cells, was examined by DNA alkaline elution with and without the use of proteinase. The resistant line was similar to the normal line with regard to the kinetics of DNA cross-link formation and removal following treatment with cis -DDP or L-PAM. For both drugs, maximum cross-linking occurred after 6 hr; this is presumed to represent the time required for conversion of drug-DNA monoadducts to cross-links. In the resistant line, interstrand cross-linking by cis -DDP or L-PAM and DNA-protein cross-linking by cis -DDP were all reduced relative to the parent line. The interstrand cross-linking was reduced in approximately the same proportion as the cytotoxicity (in terms of dose modification factors). DNA-protein cross-linking by L-PAM, however, was similar in the two cell lines. The relationship between DNA cross-linking and cell killing by cis -DDP and L-PAM was also studied in mice bearing sensitive and resistant lines of L1210 leukemia. The cells were removed from untreated mice and tested in vitro for DNA cross-linking produced by the two drugs. Tumor sensitivity was assessed by comparing the survival of treated versus untreated mice which had been inoculated with the same cells used in cross-linking assays. A L1210 line which had been developed for resistance to cis -DDP exhibited marked reductions in both types of cross-linking by this drug when compared to its sensitive parent line. This line was not resistant to L-PAM and exhibited no significant depression in cross-linking by this drug. A second line, made resistant to L-PAM, showed marked reductions in L-PAM-induced cross-linking compared to its parent line. This line was cross-resistant to cis -DDP but showed only a modest reduction in cis -DDP-induced cross-linking. Thus, in three of the four cell-drug comparisons, DNA cross-linking and in vivo cell killing were well correlated. The reason for the deviation of the fourth case was investigated in preliminary studies, but no definitive answer was obtained. The results suggest that DNA cross-linking correlates with tumor sensitivity to bifunctional agents.

Analysis of the Clinical and Biological Significance of Lymphoid Phenotypes in Acute Leukemia

Abstract: Analysis of leukemic cell phenotypes using cell surface antigens and various enzymes indicates that acute lymphoblastic leukemia (ALL) is a biologically heterogeneous disease consisting of four major subclasses with additional subsets existing within these subclasses. These different types of ALL appear to reflect sequential stages of early lymphocyte ontogeny. There is a strong association between cell phenotype and first remission duration in ALL (p trend It is suggested, therefore, that an immunological (and enzymatic) phenotype of ALL subclasses may not be an independent correlate of prognosis but nevertheless is linked to other differentiation-linked features, especially growth rate and sites of clonal expansion (e.g., marrow versus thymus), which critically influence the size of the clonogenic leukemic population and its associated evolutionary status with respect to drug-resistant mutants at the time of diagnosis and introduction of therapy. An extensive library of monoclonal antibodies has been used to further define the phenotypic heterogeneity of T- and non-T ALL. Several of the antigenic structures identified by these monoclonal antibodies have been isolated and characterized. T-ALL can be dissected into several subsets corresponding to stages of intrathymic differentiation. Non-T ALL (null-ALL, common ALL, and B-ALL) all have a phenotype indicative of B-lineage affiliation indicating that “non-T, non-B” ALL may originate from B-cell precursors in bone marrow. A cell type is identified in normal bone marrow which has the same identical monoclonal antibody-defined phenotype as common ALL and may provide the target cell for this disease.

Diet and Excretion of Bile Acids

Abstract: In recent years, salient advances have taken place in the knowledge of the interaction of diets containing high-fat and certain type of fibers and the production of bile acids potentially relevant in the etiology of colon cancer. Other studies also indicate that a high intake of certain dietary fibers, in spite of high dietary fat, not only leads to an increase in stool bulk, thus diluting carcinogens and promoters in the gut, but also modifies the metabolism of these putative substances. These studies thus suggest that both high intake of total fat and low intake of certain fibers may be necessary for the full expression of risk to colon cancer.

Carcinogenic Tobacco-specific N-Nitrosamines in Snuff and in the Saliva of Snuff Dippers

Abstract: Human data indicate an increased risk for cancer of the oral cavity for snuff dippers. Popular snuff products from the United States, Germany, Sweden, and Denmark were analyzed for tobacco-specific N-nitrosamines (TSNA). These compounds are formed during tobacco processing from nicotine, nornicotine, and anatabine and represent the only known carcinogens in snuff. N'-Nitrosonornicotine, a moderately active carcinogen, ranged in dry snuff from 3.5 to 77 ppm; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a relatively strong carcinogen, ranged from 0.6 to 7.0 ppm; and N'-nitrosoanatabine, thus far not bioassayed, ranged from 0.8 to 44 ppm. The concentrations of TSNA in a given snuff product can vary widely, and aging in the open air can lead to an increase in TSNA. Analysis of the saliva of snuff dippers revealed that these nitrosamines are extracted from the tobacco plug during snuff dipping and that their concentrations in saliva can vary widely between users. Efforts should be made to reduce the TSNA in snuff by modifications of the production process and by wrapping individual snuff portions in airtight packets.

Clonal Growth of Epithelial Cells from Normal Adult Human Bronchus

Abstract: Normal primary epithelial cell cultures devoid of fibroblastic cells have been developed from tissue explants of adult human bronchi. Conditions for clonal growth of secondary cultures of bronchial epithelial cells were optimized by coculturing the human cells with mitomycin C growth-arrested Swiss 3T3 mouse feeder cells, lowering the calcium concentration of medium M199, and supplementing it with hydrocortisone, insulin, cholera toxin, epidermal growth factor, and 1.25% fetal bovine serum. The epithelial cells grew for an average of 35 population doublings and had the normal human karyotype, expressed keratin and blood group antigen epithelial cell markers, metabolized benzo(a)pyrene, and were capable of differentiating into both ciliated and squamous cells. This culture system makes it potentially possible to investigate various aspects of differentiation and carcinogenesis in human bronchial epithelial cells.