Neoplastic conversion of human keratinocytes by adenovirus 12-SV40 virus and chemical carcinogens.

Efforts to investigate the progression of events that lead human cells of epithelial origin to become neoplastic in response to carcinogenic agents have been aided by the development of tissue culture systems for propagation of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus 12 and simian virus 40 (Ad 12-SV40) were transformed by treatment with the chemical carcinogens N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide. Such transformants showed morphological alterations and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. This in vitro system may be useful in assessing environmental carcinogens for human epithelial cells and in detecting new human oncogenes.

Neoplastic transformation of human epidermal keratinocytes by AD12-SV40 and Kirsten sarcoma viruses.

Recent investigations have begun to dissect the number and nature of genetic alterations associated with cancer cells. In the present study, primary human epidermal keratinocytes acquired indefinite life-span in culture but did not undergo malignant conversion in response to infection with a hybrid of adenovirus 12 and simian virus 40. Addition of Kirsten murine sarcoma virus, which contains a K-ras oncogene, to these cells induced morphological alterations associated with the acquisition of neoplastic properties. These findings demonstrate the malignant transformation of human primary epithelial cells in culture and support a multiple-step process for neoplastic conversion.

Inhibition of chemically induced carcinogenesis of canine cells in vitro by infection with mouse xenotropic type C virus.

C57L mouse xenotropic type C virus infection inhibited N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced in vitro transformation of embryo cells from inbred beagles. Treatment with MNNG induced in vitro neoplastic transformation in uninfected canine cells but not in C57L virus-infected canine cells; the C57L virus-infected canine cells not treated with MNNG also remained untransformed. In this dog cell system, preinfection with a C57L mouse xenotropic type C virus inhibited in vitro neoplastic transformation induced by MNNG.

Tumor progression in nude mice and its representation in cell culture.

Varying dilutions containing from 10(6) to 10(3) spontaneously transformed Balb/3T3 cells were inoculated into nude mice [N:NIH(S)II]. Less than half the mice inoculated with 10(3) cells developed tumors. The higher concentrations of cells produced visible tumors in all mice within 2-3 weeks, and these tumors grew rapidly to large sizes. Some tumors initiated by the lower concentrations of cells arose quickly, but others were greatly delayed in onset, then grew slowly, if at all, for several weeks before a rapid acceleration. The delayed acceleration can be considered a form of tumor progression. When first explanted into culture, cells from the early tumors multiplied somewhat more slowly than the parental cells that initiated the tumors, but narrowed the gap in a few weekly passages. By contrast, only a small fraction (less than or equal to 0.001) of cells from the longest delayed tumors could sustain multiplication in culture, although flow cytometry revealed them to have been a rapidly multiplying population when explanted. A relatively large fraction of these explanted tumor cells incorporated a 1-hour pulse of [3H]thymidine into DNA, although at a low rate. The shift to culture apparently slowed progress through the S-period of the cell cycle. The multiplication rate of cell populations from the delayed tumors increased in successive passages in culture. There was great heterogeneity in growth capacity among clones of the tumor cells. The growth rates of some clones declined to the point of extinction, those of others remained constant for several weeks, while those of still others steadily increased in growth rate. The low initial cloning efficiency of cells from the delayed tumors and the heterogeneity of growth rates among the clonable cells indicate that selection plays a major role in the increase of the growth capacity of the cell population. The steady increase in growth rates within clones suggests that physiological adaptation also contributes to the progressive growth of the tumor populations in culture. The results constitute a rationale for using the progressive growth of cells in culture as a model system for discriminating the types of cellular changes that underlie tumor progression.

Heritable variations in growth potential and morphology within a clone of Balb/3T3 cells and their relation to tumor formation.

A nontransformed clone and a spontaneously transformed clone were isolated from a twice-recloned line of Balb/3T3 cells. At different times two sublines were initiated from the nontransformed clone, and three were initiated from the transformed clone. The sublines were maintained in parallel passages under the same conditions. Each subline was distinctive in appearance and fell into the same rank order in a variety of growth parameters in vitro. Colony formation in agar and tumor formation in mice occurred only in the morphologically transformed sublines, but there was no quantitative correlation between the two properties or with the rate of glucose utilization. Two of the cell populations derived from noninbred NIH nude mouse tumors of the 3 transformed sublines differed in agar colony formation from the parental sublines. The results indicate that there is an immense capacity for variation in cultured animal cells involving many unrelated characteristics expressed in a way that is difficult, if not impossible, to explain by conventional genetic models.

Induction of lymphoma and associated xenotropic type C virus in C57L mice by whole-body irradiation.

Adult C57L mice received sublethal whole-body X-irradiation. Between 3 and 11 months later, 5 of the 7 exposed mice developed histopathologically confirmed malignant lymphomas (lymphocytic type) that primarily involved the thymus. The lymphomas were readily transplantable to other C57L mice of any age, which developed fetal lymphomatous involvement; the tumor cells could also be propagated in tissue culture. A xenotropic murine type C virus (MuX) was isolated from the cultured lymphoma cells after cocultivation with a permissive dog line. MuX activity was demonstrated by electron microscopy, complement fixation, indirect fluorescent antibody, infectivity, and genome rescue.

Type-C RNA viruses of the NIH nude mouse.

Several types of tumors from various species were propagated in NIH athymic nude mice. Subsequently, cell lines were established from the tumors and examined for evidence of type-C virus activity. Hybrid mice (NIH Swiss and BALB/c) harbored murine type-C viruses of three categories: N-tropic, B-tropic and X-tropic.

Human bladder carcinoma: characterization of two new tumor cell lines and search for tumor viruses.

Two newly established human bladder carcinoma cell lines, designated HT-1197 and HT-1376, were characterized. Cells of both cultures exhibited fine structural microvilli and tonofibrils indicative of their epithelial origin. In addition, desmosomes were also present in HT-1197. Marker chromosomes present in HT-1197 and HT-1376 distinguished these from each other and from other known human tumor cell lines. Both cultures grew in soft agar, induced fibrinolytic activity, and were tumorigenic in mice and hamsters. No type C or other virus expression was detected in these cell lines nor in other human urothelial tumors tested.

Proliferation of infected lymphoid precursors before Moloney murine leukemia virus-induced T-cell lymphoma.

NFS/N mice inoculated with Moloney murine leukemia virus (M-MuLV) developed T-cell lymphoma after a 10-week latent period. Expression of lymphoid differentiation antigens, appearance of M-MuLV-encoded cell surface antigens, and rates of cellular proliferation were measured in splenic and bone marrow subpopulations during this latent period. At 2 weeks of age, Thy-1-and surface immunoglobulin-negative null cells of spleen and bone marrow expressed M-MuLV antigens whereas T- and B-lymphocytes did not. During the 3d and 4th weeks, the number of splenic null cells increased to six times the number found in uninfected controls. These null cells included the precursors of lymphocytes and hematopoietic cells. For the remainder of the latent period, the percentage of null cells undergoing proliferation was three times greater in the infected mice, while the total number of null cells remained constant. This proliferation was not accompanied by terminal differentiation or emigration of mature cell types from the spleen. Proliferation was substantially delayed in CBA mice, which are resistant to lymphoma induction.

Transformation of human osteosarcoma cells by a chemical carcinogen.

A human osteosarcoma clonal cell line (TE-85, clone F-5) was treated in vitro with various levels of 7,12-dimethylbenz[a]anthracene or dimethyl sulfoxide (control). Cells treated only with the carcinogen underwent morphologic alteration in vitro, and one of these altered cell lines produced tumors subcutaneously and intracerebrally when injected into NIH nude mice.

Selection and adaptation for rapid growth in culture of cells from delayed sarcomas in nude mice.

More than 1000 cells of a spontaneously transformed line of BALB/3T3 cells are required to initiate tumors in half the nude mice inoculated s.c., although the cells clone with an efficiency approaching 100% in culture. The cells of two tumors with prolonged latent periods, initiated by 2 X 10(4) and 5 X 10(3) cells, were chosen for detailed clonal analysis in culture. The cells from the tumors grew very poorly in culture in the first passages, but with increasing speed and efficiency in later passages. Cells derived directly from the two tumors cloned in agar with an efficiency of 0.01 and 0.002%. The growth rates on plastic of the rare successful clones derived from agar were generally low but extremely varied. Some clones lost the capacity for multiplication in a few passages, while others persisted but fluctuated unpredictably in growth rate in the early passages. The graded increase in growth rate of the uncloned tumor cell populations was probably the result of selection of the more rapidly growing clones. One of the slower-growing clones was subcloned. About half of the subclones grew at a slower rate than the parental clone. These, however, increased progressively in growth rate over six successive weekly passages, suggesting the occurrence of a gradual physiological adaptation. We conclude that selection of fast-growing clones contributes a major part of the gradually improving growth of tumor cell populations in culture, but that a physiological adaptation extending over many cell generations makes a significant contribution. The mechanism in either case is unknown, and indeed there may not be a unique mechanism in the scientifically rigorous sense.

Dynamics of tumor growth and cellular adaptation after inoculation into nude mice of varying numbers of transformed 3T3 cells and of readaptation to culture of the tumor cells.

Decimal dilutions containing 5 X 10(5) to 5 X 10(1) cells were inoculated s.c. into nude mice and the course of tumor development was recorded. The highest concentration of cells produced rapidly growing poorly differentiated sarcomas within 2-3 weeks of their inoculation. Upon explantation the resultant tumors yielded cells which multiplied on plastic almost as rapidly as did their progenitors used to initiate the tumors, and had as high a colony forming efficiency in agar as long as tryptose phosphate broth was omitted from the agar medium. Tumors initiated by the lower concentrations of cells were disproportionately delayed in their appearance and tended to increase in size at a low rate. At least one tumor regressed and one which apparently regressed appeared again at a later time. These changes are characteristically described under the rubric of tumor regression. Host reactive cells such as neutrophils, eosinophils, macrophages, and fibroblasts were observed in some tumors. One of the tumors was a low grade hemangiosarcoma, another a well-differentiated fibrosarcoma, and the rest poorly differentiated sarcomas. Cells from two tumors initiated by 500 and 5000 cells multiplied slowly in early passages in culture, particularly when seeded at low densities at which they appeared to sustain cumulative damage even when multiplying. In later passages, the "low dose" tumor cells gained the capacity to multiply in culture after seeding at low densities, but it took up to 50 cell generations to reach this capacity. The loss of growth capacity on plastic of cells from the low dose tumors and its subsequent restoration by passaging in culture may provide a quantitative method for analyzing the type of cellular change which underlies tumor progression.

High-frequency variation and population drift in a newly transformed clone of BALB/3T3 cells.

During repeated passage of BALB/3T3 cells and testing for anchorage-independent growth, a single transformed clone was isolated from agar, and five subclones were derived from it. These subclones differed from one another in morphology on a solid substratum, efficiency and size of colony formation in agar, and rate of tumor formation in nude mice. With weekly passage over a period of 6 months, the differences in morphology and growth in agar gradually decreased. The subclone which produced the fastest-growing tumors in nude mice after 4 weeks of culture produced the slowest-growing tumors after 18 weeks, and a change in the opposite direction was made by another subclone. There was no difference among the subclones in growth rate on plastic. The distribution of chromosome numbers was heterogeneous but overlapping in all the primary subclones at 16 and 24 weeks, with no statistically significant difference in the mean number of chromosomes per subclone. An extremely high degree of variation must have occurred to produce the multiple differences between the subclones, and the same type of variation could have been responsible for the subsequent changes with repeated passage. The high frequency and graded nature of the changes and the concurrent involvement of several traits suggest an epigenetic basis for the variation.

Inheritance of acquired changes in growth capacity of spontaneously transformed BALB/3T3 cells propagated in mice and in culture.

Five subclones were derived from a spontaneously transformed BALB/3T3 clone soon after its isolation. Despite their common clonal origin, the subclones were different from each other in appearance, colony-forming efficiency in agar (CFEag), and rate of tumor formation in mice. A comparison of growth properties during repeated passages in culture was made between the cells derived from the tumors and the cells used to initiate the tumors. In most cases, the tumor-derived cells had a much lower CFEag than did their parental in vitro-propagated cells, and the CFEag was restored slowly to the original level or remained at a reduced level during the period of study. In a few cases, the tumor-derived cells had almost as high a CFEag as their parental cells or were quickly restored to this level during cultivation. It was shown by karyotypic and clonal analysis that the reduced CFEag of the tumor-derived cells arose from a change in the transformed cells; i.e., it was not due to the presence of normal host cells in the explanted tumor. Clones of the tumor-derived cells tended to show the same patterns of change in CFEag as the uncloned tumor cell populations, but there were cases of individual variation in pattern among the tumor cell clones. Tumor cells with greatly reduced CFEag also grew a little more slowly on plastic than did the parental nontumor cells, and their growth rate tended to increase along with CFEag in long-term culture. Clonal analysis of one of the five original subclones failed to reveal cells which had the CFEag properties of its progeny tumor cells. This suggests that the reduction of CFEag during tumor formation arose by adaptation rather than selection of preexisting variants. A similar conclusion was drawn about the restoration of CFEag during cultivation of the tumor-derived cells. Although the decrease in CFEag which accompanied tumor formation varied in magnitude and stability, some tumor cell populations retained their reduced capacity through months of weekly passaging in culture, involving up to 100 cell divisions. The results are therefore consistent with the heretical notion of inheritance of acquired characteristics. In addition, the wide variation of in vitro growth capacity among tumors initiated by different subclones, and even among tumors initiated by the same subclone, raises the possibility that the complete chain of causality underlying the variation is intrinsically indeterminate.

Neoplastic transformation of a human keratinocyte cell line by the v-fos oncogene.

To analyze the role of the fos oncogene in the growth of human epithelial cells, we have transfected a non-tumorigenic human epidermal keratinocyte line (RHEK-1) immortalized by the Ad12-SV40 hybrid virus with a plasmid carrying the v-fos gene together with plasmid pSV2-neo which confers resistance to neomycin. Individual neomycin-resistant clones were isolated and characterized with respect to morphological alteration. Of 16 independent clones analyzed, two appeared morphologically transformed and formed foci in culture. Only the two clones with a transformed phenotype were found by Southern blot hybridization analysis to contain the transfected v-fos gene. These clones formed colonies in soft agar and induced tumors when transplanted into nude mice. Analysis of fos specific mRNA and protein demonstrated that the transfected v-fos gene was expressed in these two clonal lines. These findings suggest that expression of the v-fos gene might facilitate the process of neoplastic transformation of human epithelial cells in culture. This appears to represent the first demonstration of the transforming potential of the v-fos gene in human cells.

Oncogenic potential of erbB-2 in human mammary epithelial cells.

Introduction of the normal erbB-2 gene into immortalized human mammary epithelial cells (184B5) by transfection conferred a growth advantage to these cells both in vitro and in vivo. The 184B5 cells overexpressing erbB-2 formed colonies in semi-solid medium, frequently induced transient nodules in athymic mice and produced progressive tumors in vivo at a low frequency. Those tumors which did arise from erbB-2-transfected cells displayed substantially higher levels of normal gp185erb-2 protein when compared to the original transfectants, consistent with their selection for increased erbB-2 expression. Introduction of genes encoding genetically altered erbB-2 molecules into 184B5 cells increased their colony-forming efficiency and converted the cells to a tumorigenic phenotype at a high frequency. When the biological and biochemical properties of human mammary carcinoma cell lines known to overexpress erbB-2 were compared to the transfected 184B5 lines, they behaved most like those overexpressing the normal erbB-2 protein. Results indicate that overexpression of normal erbB-2 may directly contribute to the transformation of human mammary epithelium if sufficient levels of erbB-2 protein are expressed or if the erbB-2 gene is genetically altered.

Cooperation of V-oncogenes in human epithelial cell transformation.

The development of tissue culture systems for propagation of human epithelial cells has aided the investigation of events that lead epithelial cells to become neoplastic. In the present study, nontumorigenic human epidermal keratinocytes, immortalized by Ad12-SV40 virus or pSv3-neo, were transformed by a variety of retroviruses containing bas, H-ras, fes, fms, erbB and src oncogenes. Such transformants showed morphological alterations and induced carcinomas when transplanted into nude mice. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of Ad12-SV40 virus and retroviral oncogenes and support a multistep process for neoplastic conversion. This in vitro system may be useful in studying the interaction of a variety of retroviral oncogenes and human epithelial cells.

Preoperative management of the amputated limb.

Little literature exists on storage and preparation of an amputated limb prior to transfer to a reconstructive plastic surgical unit for possible macroreplantation. This paper describes practical measures used to prolong ischaemia time allowing macroreplantation, tissue harvesting, or fashioning of a useful stump. A simple protocol is used to summarise these points.

Characterization of a human Kaposi's sarcoma cell line that induces angiogenic tumors in animals.

OBJECTIVE: To characterize a Kaposi's sarcoma (KS) cell line established from a tumor biopsy from the oral mucosa of an iatrogenically immunosuppressed HIV-negative man. METHODS: Cells were placed in culture and evaluated by a variety of biologic, serologic, karyotypic, and immunologic procedures. Electron microscopic examination was performed. The ability to produce tumors in nude mice was evaluated, and the nature of the cells within the tumor determined. Assays for urokinase plasminogen activator type (uPA), plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR) were conducted. RESULTS: The SLK cell line has an endothelial cell morphology with very little anaplasia. The karyotype indicates diploid phenotype of human origin. Immunohistochemical and electron microscopic examinations confirmed the endothelial nature of this cell line. No viruses were detected. The tumors induced in nude mice showed hypervascularization, with characteristics of KS. The cell line produces uPA and PAI-1, and also expresses uPAR. CONCLUSIONS: The SLK cell line is of endothelial cell origin and the first human cell line to induce KS-like tumors in recipient animals. The expression of urokinase and its receptor suggests a paracrine and autocrine interaction that may be important for the growth of the tumor. The SLK line should be valuable for studies of KS pathogenesis and therapeutic approaches to this malignancy.

Predicting completion of a cognitive-behavioral pain management program by initial measures of a chronic pain patient' s readiness for change.

OBJECTIVE: There is a need to identify pretreatment patient indicators, which are predictive of the successful enrollment and completion of chronic pain treatment programs. Recent evidence suggests the Pain Stages of Change Questionnaire can predict enrollment and completion of a 10-session cognitive-behavioral pain management program. The purpose of this study is to determine whether the pretreatment Stages of Change Questionnaire can predict patients who would complete a cognitive-behavioral pain treatment program. DESIGN: Prospective cohort study using logistic regression analyses. SETTINGS: Patients referred for a 10-session cognitive-behavioral treatment program at a tertiary care multidisciplinary pain clinic or a community-based specialty clinic. SUBJECTS: Three hundred chronic pain patients (151 in the tertiary setting and 149 in the community-based setting) participated, with 147 of the patients (49%) completing and 153 (51%) patients not completing the 10-session program. INTERVENTION: Ten-visit cognitive-behavioral program for chronic pain patients. OUTCOME MEASURE: Completion of program. RESULTS: The Stages of Change Questionnaire scores could predict completion status chi2 (N = 300, 2 df) = 39.7, p <0.001, (goodness-of-fit test chi2 = 5.69, p = 0.68). Those patients completing the program were slightly older and reported higher levels of pain, depression, and disability than did those patients who did not complete. Low "Precontemplation" score remained the best single predictor, as it identified correctly 61% of the cases patients who completed the program and predicted who would drop out in 65% of the cases. CONCLUSION: The Stages of Change Questionnaire is a potentially useful tool; however, the current scoring method is insufficient to recommend its use as an inclusion or exclusion criterion for enrollment in a cognitive-behavioral program.