Cell-cell contact interactions conditionally determine suppression and selection of the neoplastic phenotype

Separation of chemical and physical carcinogenesis into the stages of initiation (mutation) and promotion (selection) established that incipient neoplastic cells could persist in the organism indefinitely without expression. Spontaneous mutations associated with cancer also lie dormant in untreated normal tissue. Without selection, there is no tumor development. Experiments in cell culture showed that confluent normal fibroblasts suppress growth of contacting transformed fibroblasts, and that normal keratinocytes similarly suppress tumor formation by adjacent papilloma cells. With cells that are generally more susceptible to transformation, however, prolonged contact inhibition progressively selects mutants that favor neoplastic growth. Selection of individual mutant cells allows them to become a significant fraction of the population and creates an enlarged target for additional genetic hits. Crucially, this enrichment step, not the initial mutation step, is the numerically limiting factor in tumor development. Unexpectedly, variants that are resistant to spontaneous transformation are selected in vitro by growing cells for many low density passages at maximal exponential rate. Confluent cultures of resistant variants suppress the growth and normalize the morphology of contacting transformed cells. Varying the conditions for selection shows that tumorigenic transformation is preceded by intermediate steps of progressively higher saturation density that are increasingly permissive for the expression of the more neoplastic cells in the population. There is also evidence of increasing permissiveness with age of normal tissues in vivo for solitary cancer cells transplanted in their midst. Spontaneous transformation in culture can be used to identify dietary components that are required for promotion and may therefore be applicable in prevention of human cancer.     

Characterization of a new non-Hodgkin's lymphoma cell line (NCEB-1) with a chromosomal (11:14) translocation [t(11:14)(q13;q32)]

A new cell line, NCEB-1, was established by Epstein-Barr virus (EBV) transformation of peripheral blood mononuclear cells from a patient with centroblastic-centrocytic diffuse lymphoma expressing IgM lambda. The transformed cells were lymphoblastoid, with many cells showing a plasmacytoid morphology. The NCEB-1 cells had cytoplasmic Ig (CyIg), with loss of the surface Ig (SIg) expression. Cytogenetic analysis of the cell line demonstrated two clones with variations: a hypodiploid clone, with a complex karyotype including a t(11;14)(q13;q32) similar to the original tumor cells, and a near tetraploid clone with the same markers. Southern blot analysis of DNA from the patient's neoplastic cells and NCEB-1 demonstrated identical Ig heavy chain gene rearrangement, confirming the origin of the cell line. The cell line was not tumorigenic when tested in an in vitro assay using immunosuppressed mice. NCEB-1 has been in continuous culture for 9 months and will be valuable for the in vivo study of non-Hodgkin's lymphoma and EBV transformation.     

HIV type 1 Nef promotes neoplastic transformation of immortalized neural cells

To study the effects of HIV-1 Nef on CNS-derived cells in vivo, an expression system based on the murine neural stem cell line C17.2 was established. Stable expression of LAV-1(Bru)-nef in these cells induced a transformed phenotype and enhanced cell growth in soft agar. Further experiments using previously established nef-expressing human astrocytoma cell lines as well as nef-expressing murine fibroblasts suggested a brain cell-specific transforming activity of Nef. After implantation into syngeneic or nude mice both murine and human nef-expressing CNS-derived cells induced tumor development. Interestingly, human astrocytoma cells expressing a Nef mutant carrying a disrupted SH3-binding motif involved in protein-protein interactions failed to induce tumor formation. These in vivo data suggest that Nef promotes neoplastic transformation of immortalized murine neural stem cells and enhances malignancy of low-tumorigenic human astrocytoma cells. Nef may therefore be involved in the development of AIDS-associated brain tumors.     

Neoplastic transformation of immortalized human epidermal keratinocytes by ionizing radiation.

Efforts to investigate the progression of events that cause human cells to become neoplastic in response to ionizing radiation have been aided by the development of tissue culture systems of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus type 12 and simian virus 40 have been transformed by exposure to x-ray irradiation. Such transformants showed morphological alterations, formed colonies in soft agar, and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes exposed to radiation in this manner failed to show any evidence of transformation. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of a DNA tumor virus and radiation, indicating a multistep process for radiation-induced neoplastic conversion. This in vitro system may be useful as a tool for dissecting the process of radiation-induced neoplastic transformation of human epithelial cells and for detecting previously unreported human oncogenes.     

Promise and problems in relating cellular senescence in vitro to aging in vivo

According to the 'Hayflick limit', human fetal fibroblasts have a uniform, limited replicative lifespan of about 50 population doublings in cell culture. This concept was extrapolated to diverse cells in the body. It seemed to decrease with the age of the cell donor and, as a form of cell senescence, was thought to underlie the aging process. More discriminating analysis, however, showed that the fibroblasts decayed in a stochastic manner from the time of their explantation, at a rate that increased with the number of population doublings in culture. There was no consistent relation to the age of the donor. Despite the contradictory evidence, the original version of the Hayflick limit retained its general acceptance. Cell senescence was attributed to the absence of telomerase in the fibroblasts, which resulted in shortening of telomeres at each division until they fell below a critical length needed for further division. However, it is well established that stem cells in renewing tissues undergo many more than 50 divisions in a lifetime, without apparent senescence. Contrary to early findings of no telomerase in most tissues, their stem cells retain telomerase and presumably telomere length despite many divisions in vivo. Massive accumulation of lipofuscin granules occurs under stress in long term crowded cultures, but the granules dissipate on subculture or neoplastic transformation. The overall results indicate a critical disjunction between cell senescence in vitro and aging in vivo. By contrast, cell culture has been useful in showing a need for telomere capping in maintaining cell stability and viability. It may also provide information about the biochemical mechanism of lipofuscin production.     

Parathyroid hormone-related protein expression and secretion in a skin organotypic culture system

Parathyroid hormone-related protein (PTHrP), an important factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many normal tissues, including the epidermis, where it is thought to play a role in the regulation of keratinocyte growth and differentiation. Most in vitro studies of normal keratinocytes use monolayer cell cultures, which have limitations, including the inability to reproduce the stratified structure of the epidermis. The objective of this study was to investigate PTHrP production and secretion, and mRNA expression in skin organotypic cultures. The cultures consisted of an artificial dermis with differentiating keratinocytes grown at the air-liquid interface. Immunohistochemical assessment of cytokeratins 14 and 10/13, involucrin, and proliferative cell nuclear antigen (PCNA) demonstrated that keratinocytes differentiated in a manner similar to keratinocytes in normal epidermis. PTHrP expression was demonstrated in all viable layers of the epidermis, as well as in some fibroblasts of the collagen lattice by immunohistochemistry and in situ hybridization. Since most fibroblasts expressed α-smooth muscle actin, these cells were interpreted to be consistent with myofibroblasts. PTHrP expression by myofibroblasts suggests a possible role for PTHrP in the regulation of contractibility of these cells. PTHrP was also detected in conditioned media for 50 days. In conclusion, because of its superior tissue morphology and ability to induce organized keratinocyte differentiation, this culture system will be an excellent model to study the role of PTHrP in pathologic and physiologic processes involving the epidermis in vitro.     

Parathyroid hormone stimulates the proliferation of cells derived from human bone

Despite its acute inhibitory effect on bone formation in vitro, PTH has been shown to have an anabolic effect on bone in vivo and to stimulate cell proliferation in osteoblastic cell lines and organ cultures. We have examined the effects of PTH on cells derived from human trabecular bone and compared these effects with those on human skin fibroblasts. Human bone cells have the capacity to synthesize type I collagen and osteocalcin, and to respond to 1,25-dihydroxyvitamin D3 with an increase in the synthesis of osteocalcin and alkaline phosphatase. PTH stimulated adenylate cyclase activity at both low and high cell density. However, the same concentrations of hormone stimulated the proliferation of these cells only when they were cultured at a high cell density. The effect of PTH was bone cell specific in that no proliferative effect of PTH was detected in cultures of human skin fibroblasts obtained from the same donor and cultured under the same conditions. The effect of PTH on DNA synthesis by human bone cells may be important in the generation of a long term anabolic response to PTH.     

Hepatitis B virus and hepatocellular carcinoma: molecular biology and mechanistic considerations

The question of whether HBV causes HCC has to date best been answered by epidemiologic studies, in particular prospective analysis such as the ongoing study in Taiwan. The mechanism whereby HBV may cause HCC by genetic or epigenetic means, acting alone or in concert with other agents, is still moot . To date, studies of HBV gene expression have not shown the presence of a transforming gene or an oncogene associated with this virus. The finding of integrated HBV DNA in most HCC of carriers is highly suggestive of a causal role of the virus in the pathogenesis of neoplasia. In order to be more certain, it will be necessary to show that expression of integrated HBV genes or of cellular genes, as a result of HBV infection, causes neoplastic transformation of cells. Failure to date to demonstrate that HBV DNA induces neoplastic transformation of eukaryotic cells in culture may be simply due to an inadequate experimental model. The majority of these experiments have used fibroblasts or monkey kidney cells as the recipient cell, whereas cultured human hepatocytes might be more suitable. Whatever role HBV eventually is shown to have in the pathogenesis of HCC, molecular biologic techniques will have in great measure contributed to that knowledge. The use of these powerful new methods will permit studies of the replication of HBV and possible elucidation of how this virus affects the hepatocyte during persistent infection and will in time lead to a better understanding of cell and molecular events leading to development of hepatic malignancy.     

T-lymphocyte long-term cultures have a constant histone variant pattern during aging

Human peripheral long-term T-lymphocyte cell cultures show some characteristics similar to those of fibroblast cell lines, the latter of which have been used as in vitro systems for cellular aging studies for many years. Both show a limited in vitro life span, as well as a progressive prolongation of their cell cycle with increasing age. However, whereas T-cell cultures die from apoptosis at the end of their proliferative capacity, fibroblasts can be maintained for long periods of time in stationary cultures as postmitotic senescent cells. Previous studies analyzing the histone variant pattern of a human lung embryonic fibroblast cell line have shown that this pattern changes as a function of cumulative population doublings in a manner not unlike that found in terminally differentiating systems. In the present study the histone variant composition of long-term T-cell cultures was analyzed as a function of population doublings and compared to a human diploid fibroblast system. The results from this study provide a distinction at the molecular level among these two in vitro aging model systems, because it was found that long-term T-cell cultures show a constant histone variant constitution throughout their in vitro life, dissimilar to previous findings using the fibroblast cell system.     

Experimental evidence for a unifying concept of the molecular mechanisms of the cellular aging and the cellular neoplastic transformation.

Experimental data supporting a Unifying Concept of the molecular mechanisms of cellular aging and cellular neoplastic transformation of dividing cells in mass culture and clonal culture systems will be described. The Unifying Concept combines the hitherto antithetically presented Differentiation Theory, the Mutation Theory, the Error Catastrophy Theory and the Degradation Deficiency Theory with a newly worked out Virus Theory of the cellular aging and the cellular neoplastic transformation. Quantitative in vitro studies of embryonic fibroblast cell systems of two closely related inbred rat strains L.BN and Lewis were undertaken. The data obtained from the experimental analysis of the molecular mechanisms of the cellular aging and the cellular neoplastic transformation demonstrate, that the cellular aging of dividing cells is a Three-Stage-Differentiation Sequence under the control of three different genetic programs. The genetic constitution of the senescent cell regulates the expression of virogenes and oncogenes of the endogenous RNA tumor viruses of the C-type, resulting either in the cellular degeneration of the senescent cell under the control of the virogenes or in the cellular neoplastic transformation regulated by the oncogenes.     

Cell culture systems to study progression and inhibition of intimal proliferations

Summary Vascular smooth muscle cells, endothelial cells, and fibroblasts are the main cellular constituents of artery walls. Mass cultures and clone cultures of these cell types have meanwhile become valuable tools in the research of the genesis, pathophysiology, and therapy of vessel-wall diseases. With transfilter co-culture systems the three-layered construction of the artery wall can be imitated in vitro, and it has become possible to induce smooth muscle cell proliferates in these in vitro system which resemble, in many respects, intimal proliferates as they often occur after angioplasty, stent- or bypass operations in the form of secondary stenoses. With this technique the interaction of the three cell species of artery walls can be easily studied. The time-course of the development of smooth muscle cell proliferates in vitro resembles the in vivo scenario. Addition of oxidized lipoproteins and monocytes to the culture medium of transfilter cultures leads to atheroma-like proliferates. Culturing whole artery segments is another in vitro technique for induction of intimal proliferates, and enables the production of intimal proliferates in a way similar to transfilter culture systems. Because of the striking similarities of the cellular responses of transfilter- and organ-culture systems with in vivo processes in atherogenesis, and in the development of secondary stenoses after angioplasty, the described co-culture systems are suitable for studying the genesis, pathophysiology, and therapy of stenosing artery processes, as well as to obtain furthrt insight into basic problems of cell interaction in vessel walls.     

Conditions for inhibiting and enhancing effects of the protease inhibitor antipain on x-ray-induced neoplastic transformation in hamster and mouse cells.

Using cultured normal hamster embryo cells and the heterploid mouse C3H cell line 10T1/2, clone 8, we have studied the effect of the protease inhibitor antipain on x-ray-induced neoplastic transformation. We found in both cell systems that, while there was no effect on cell survival as compared to irradiated controls, the addition of antipain at a concentration of 6 microgram/ml to the cultures 24 hr prior to irradiation resulted in enhanced transformation as compared to the frequency in cultures exposed to radiation alone. Yet the addition of antipain to cultures 10 min after irradiation resulted in a decreased transformation rate. This decrease was not found when antipain was added to the mouse cells 24 hr after irradiation or to the hamster cells 48 hr after irradiation. These results suggest that the protease inhibitor antipain has more than one mechanism of action in modulating the fixation and expression of transformation by x-irradiation, possibly by the modification of DNA repair.     

Cellular aging, destabilization, and cancer.

Three major characteristics of aging in animals are a slowdown of cell proliferation, an increase in residual bodies associated with age pigments, and a marked increase in the likelihood of neoplastic transformation. The 28 L subline of the NIH 3T3 line of mouse embryo fibroblasts exhibits all these characteristics when held at confluence for extended periods. The impairment of proliferation is the first behavioral characteristic detected in low density subcultures from the confluent cultures, and it persists through many cell generations of exponential multiplication. There is an equal degree of growth impairment among replicate cultures (lineages) recovered after each of 2 successive rounds of confluence, although heterogeneity appears after the third round. The growth impairment pervades the entire cell population of each lineage. The degree and duration of impairment increase with repeated rounds of confluence. A marked increase of residual bodies characteristic of age pigments occurs in the cytoplasm of all the cells kept under prolonged confluence. Neoplastic transformation first appears as foci of multilayered cells on a monolayered background of nontransformed cells. The transformed cells arise at different times in the lineages and originate from a very small fraction of the population. The transformed cells selectively overgrow the entire population in successive rounds of confluence leading to an increase in saturation density of each lineage at different times. Under cloning conditions, isolated colonies of transformed cells develop more slowly than colonies of nontransformed cells but eventually reach a higher population density. The regularity of persistent growth impairment among the lineages and the appearance of large numbers of residual bodies in all the cells of each population are more characteristic of an epigenetic process than of specific local mutations. although random chromosomal lesions cannot be ruled out. By contrast, the low frequency and stochastic character of neoplastic transformation are consistent with a conventional genetic origin. The advent in long-term confluent NIH 3T3 cultures of three cardinal characteristics of cellular aging in vivo recommends it as a model for aging cells.     

Studies using different human cell-culture systems for demonstrating interferon induction under in vitro conditions

Five human cell culture systems (FL-cells, suspensions of tonsillar cells, FL-cells and suspensions of tonsillar cells combined, human fibroblasts, leukocyte suspensions) were tested with the international standard inducer Poly (IC) for the estimation of an in vitro induction of interferon under in vitro conditions. All induced cell systems have produced IFN. It can be concluded that alone the cell culture system represented by fibroblasts and leukocytes allow a reliable prediction concerning the induction of interferon under in vitro conditions.     

Paracrine-stimulated gene expression profile favors estradiol production in breast tumors

Paracrine interactions between adipose fibroblasts and malignant epithelial cells are essential for structural and hormonal support of breast tumors. Factors derived from malignant epithelial cells inhibit adipogenic differentiation of fibroblasts and upregulate expression of aromatase, which stimulates estrogen synthesis and creates a localized, growth-stimulatory environment. Here, we characterized the gene expression profile of breast adipose fibroblasts in an in vitro model of malignancy to identify other paracrine interactions that support tumor growth. Primary breast adipose fibroblasts from cancer-free women were treated with conditioned media from malignant breast epithelial cells or normal breast epithelial cells, and differences in gene expression were identified by microarray. A total of 79 differentially regulated genes encoding cytokines, enzymes, angiogenic factors, cytoskeletal proteins, extra-cellular matrix remodeling proteins, signal transduction proteins and cell surface receptors were identified, and 6 of these were verified by real-time PCR. Among these, the expression of aldo-keto reductase family 1, member C3 (AKR1C3) was upregulated. AKR1C3 has multiple enzymatic properties, including conversion of estrone to estradiol and androstenedione to testosterone. Immunoreactive AKR1C3 was detected in epithelial and stromal components of benign lesions and ductal carcinomas in situ, and in 59.8% of epithelial and 69.6% of stromal cells in invasive breast carcinomas. AKR1C3 expression was significantly higher in myoepithelial cells surrounding the neoplastic epithelium of ductal carcinoma in situ compared with those surrounding benign epithelial lesions. Importantly, AKR1C3 and aromatase mRNA levels correlated positively in 61 malignant breast tumors (R=0.3967, p=0.00156). Malignant epithelial cell-conditioned medium significantly increased formation of testosterone and estradiol from androstenedione in breast adipose fibroblasts. In conclusion, malignant epithelial cell-derived factors significantly upregulate the enzymes AKR1C3 and aromatase that catalyze a series of complementary reactions to convert the circulating precursor androstenedione to biologically active estradiol in vitro in the stromal fibroblasts, and in vivo, in stromal component of breast tumors.     

Transforming events in thyroid tumorigenesis and their association with follicular lesions

Thyroid tumors comprise a broad spectrum of neoplastic phenotypes, and distinct molecular events have been implicated in their pathogenesis. Pituitary tumor transforming gene, originally isolated from GH(4) pituitary cells, is tumorigenic in vivo, regulates basic fibroblast growth factor secretion, and is homologous to a securin inhibitor of chromatid separation. Pituitary tumor transforming gene 1 is expressed at low levels in several normal human tissues and is abundantly expressed in neoplasms, including colorectal carcinoma, where pituitary tumor transforming gene expression correlated highly with tumor invasiveness. As pituitary tumor transforming gene is regulated by E and as thyroid cancer shows a strong female preponderance, we examined pituitary tumor transforming gene 1 expression and action in human thyroid tumors and in normal human and rat thyroid cells. Increased pituitary tumor transforming gene 1 expression was evident early in thyroid tumors and was most abundantly expressed in a subset of thyroid hyperplasia, follicular adenomas, and follicular carcinomas (1.8-fold; P < 0.0001). Pituitary tumor transforming gene 1 overexpression in rat FRTL5 thyroid cells and in primary human thyroid cell cultures causes in vitro transformation and produces a dedifferentiated neoplastic phenotype. As pituitary tumor transforming gene 1 was abundantly overexpressed in follicular adenoma and follicular carcinoma, we propose that pituitary tumor transforming gene overexpression may play a role in the early molecular events leading to divergent development of follicular and papillary carcinoma.     

Prevention of Malignant Change in Mammalian Cells During Prolonged Culture In Vitro

Mixed cultures of epithelial cells and fibroblasts, derived from primary cultures of the skin of embryo rats, grown always in rubber-stoppered T-60 flasks, first yielded a transplantable tumor from the 52nd passage, at the end of 13 months of frequently repeated subculture. A group of subcultures, derived from the 22nd passage, grown under the same medium, with the addition of 1% oxyhemoglobin, failed to yield a tumor in 23 months of repeated subculture. A return of these cultures to the regular medium with oxyhemoglobin, yielded a tumor in 4 months, after 12 more passages. Cultures of transformed cells that had regularly yielded a transplantable tumor, for 6 years, up to the 305th passage, continued to yield transplantable tumors when 1% oxyhemoglobin was added to the medium. The cells remained highly atypical, microscopically, and there was no indication of reversal of the malignancy. Although oxyhemoglobin in the medium of cell cultures seems to have had the ability to keep malignancy in abeyance, it did not reverse the established malignant transformation of the cells.