5-Azacytidine inhibits adipocytic conversion of human skin fibroblasts by Kirsten murine sarcoma virus and dexamethasone

Transformation of human skin fibroblasts (SF) by the Kirsten murine sarcoma virus (KiMSV) is associated with their neodifferentiation into preadipose cells. Hydrocortisone or dexamethasone (DX) promote the transformation/neodifferentiation of preadipocytes to fully differentiated adipocytes. 5-Azacytidine (5-Aza-CR) by itself, or together with DX, or immediately after inoculation with KiMSV did not cause adipocytic conversion of human SF. Addition of 5-Aza-CR either before or immediately after exposure of human SF to KiMSV and DX reduced the total number of foci and significantly inhibited expression of adipocyte-containing foci. The results suggest that adipocytic conversion of human SF by KiMSV and DX is specific. This conversion apparently does not involve mechanisms directly related to the effects of 5-Aza-CR on the adipocytic conversion of murine embryo cells.     

Histiocytic conversion of human adult skin fibroblasts by the Snyder-Theilen feline sarcoma virus

Oncogenes are apparently involved in the transformation/neodifferentiation of human cells. We now report a novel example of transformation/neodifferentiation: the specific conversion of cultured human adult skin fibroblasts (HSF) to histiocytes (tissue macrophages (TM)) by the Snyder-Theilen feline sarcoma virus (ST: (FeSV)). The de novo conversion of cultured HSF was demonstrated in a large fraction of ST:FeSV (FeLV)-transformed foci in the presence of dexamethasone (DX). Identification of tissue macrophages in ST:FeSV(FeLV)-infected HSF cultures was established by light-and transmission electron microscopy, histochemistry, immunohistochemistry, adherence-reattachment, latex particle uptake, and secretion of bioactive interleukin-1. The ST:FeSV gene and glucocorticosteroids, or other naturally occurring hormones, may play a role in morphogenetic processes within cells from a variety of normal and diseased tissues in situ, including induction of non-bone marrow, mesenchyme-derived, tissue macrophages.     

Improvement of carcinogen identification in BALB/3T3 cell transformation by application of a 2-stage method

The present studies intend to heighten the sensitivity of BALB/3T3 cells to chemical carcinogens in a transformation assay, by including exposure of carcinogen-treated cells to a tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA). In the assay, cells were first treated with a known or suspected carcinogen for 72 h, cultured in normal medium for 3 days, exposed to media with and without TPA for 2 weeks, and cultured in normal medium for an additional 3 weeks. Benzo[a]pyrene, a potent carcinogen with a polycyclic aromatic hydrocarbon structure, caused transformation in the presence and absence of TPA. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), a carcinogen with direct-acting alkylating ability, did not induce significant transformation without TPA, while treatment with MNNG followed by TPA produced numerous transformed foci, classifying MNNG as an initiating agent of transformation under the condition presented in this report. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), sodium nitrite and butylated hydroxyanisole (BHA), which are carcinogenic and/or mutagenic, produced transformed foci in significant numbers of treated dishes in the presence but not in the absence of TPA. Butylated hydroxytoluene (BHT) and sodium saccharin, which are considered to be a modifier and a promoter of carcinogenesis, did not cause significant transformation with or without TPA treatment. These studies suggest that this 2-stage transformation system is capable of detecting a wider range of chemical carcinogens as initiating agents than the standard assay. Studies on the transformation assay schedule revealed that the proportion of dishes with foci, the number of foci per dish and sizes of foci all increased in the normal medium after the termination of TPA treatment. Therefore, transformed cells appear to proliferate independently of TPA after those cells are released by TPA from postconfluence inhibition of cell division.     

Increased susceptibility to SV40 transformation with development and in vitro aging

The incidence of most cancers increases with aging. To examine whether this increased risk might be related to a higher susceptibility of older cells to neoplastic transformation, we transfected rat fibroblasts aged in vivo and in vitro with origin-defective SV40 DNA and measured the number of transformed foci. Substantial increases in the number of transformed foci were observed in cells from adult rats when compared with those of cells from embryos or weanlings. Much higher numbers of foci were also obtained at late passage, when 68% or more of the in vitro lifespan had been completed, while no foci were produced from cells at early or middle passage. To control for changes with aging in uptake, integration, or expression of exogenous DNA, parallel cultures were transfected with a G418 resistance gene. The number of G418-resistant colonies did not increase with aging and, in fact, decreased in late passage embryonic cell cultures. Therefore, increased susceptibility to SV40 transformation appears to be a feature of development and in vitro aging in rat cells.     

Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40

A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC. After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40). A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far. The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure. Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line. The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts. The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules. Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies.     

Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40

Summary A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC. After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40). A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far. The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure. Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line. The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts. The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules. Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies. This work was supported by Grant 82.00346.96 from the C.N.R. finalized project “Control of Neoplastic Growth”.     

Glucocorticoids modulate transformation by v-Src in a cell-specific manner

In Balb 3T3 murine fibroblasts infected with retroviruses carrying the v-src oncogene, treatment with the glucocorticoid hormone dexamethasone induces a 10-fold increase in the number of transformed foci and of anchorage-independent colonies. In contrast, in NIH-3T3-infected cells the number of foci and of colonies growing in soft agar is considerably reduced by the addition of the hormone. The effect of dexamethasone on both Balb 3T3 and NIH 3T3 cells is dose-dependent and mediated by specific receptors. The expression of glucocorticoid receptors as well as transactivation of a mouse mammary tumor virus promoter in the presence of dexamethasone is comparable in the two cell lines. Dexamethasone does not change the expression and kinase activity of v-Src proteins either in freshly infected Balb 3T3 and NIH 3T3 cells or in morphologically normal clones or in transformed foci derived from infected Balb 3T3 cells stably expressing v-Src. However, in cocultivation assays of phenotypically normal clones of v-Src expressing Balb 3T3 cells mixed with a large excess of parental Balb 3T3 cells, the hormone is able to rescue the ability to form transformed foci of these otherwise normal cells. The present data point out a new role of glucocorticoid hormones in controlling transformation in a cell-specific manner through epigenetic mechanisms.     

Increased susceptibility to SV40 transformation with development and in vitro aging

The incidence of most cancers increases with aging. To examine whether this increased risk might be related to a higher susceptibility of older cells to neoplastic transformation, we transfected rat fibroblasts aged in vivo and in vitro with origin-defective SV40 DNA and measured the number of transformed foci. Substantial increases in the number of transformed foci were observed in cells from adult rats when compared with those of cells from embryos or weanlings. Much higher numbers of foci were also obtained at late passage, when 68% or more of the in vitro lifespan had been completed, while no foci were produced from cells at early or middle passage. To control for changes with aging in uptake, integration, or expression of exogenous DNA, parallel cultures were transfected with a G418 resistance gene. The number of G418-resistant colonies did not increase with aging and, in fact, decreased in late passage embryonic cell cultures. Therefore, increased susceptibility to SV40 transformation appears to be a feature of development and in vitro aging in rat Cells.     

Catecholamine stimulated lipolysis in differentiated human preadipocytes in a serum-free,defined medium

Adipocyte precursors from the stromal vascular fraction of human adipose tissue were allowed to differentiate in serum-free defined medium, whereafter their catecholamine stimulated lipolytic response was compared to that of mature isolated human adipocytes. Seventy-five to ninety percent of the fibroblast-like cells accumulated lipid droplets and glycerol-3-phosphate dehydrogenase activities of 1,000–2,800 mU/mg protein were measured in cell homogenates of differentiated cells. Lipolysis could be stimulated by both isoproterenol and norepinephrine in both differentiated preadipocytes as well as mature adipocytes. The results obtained with β-adrenergic agents suggested the presence of a higher affinity receptor in differentiated preadipocytes as compared to mature adipocytes. Mature adipocytes responded well to β-adrenergic agents, but no antilipolytic α₂-adrenergic response was observed in the differentiated preadipocytes. The presence of Gi proteins in the differentiated preadipocytes was suggested by the antilipolytic effect of adenosine as well as the lipolytic activity generated by pertussis toxin. In conclusion, our medium supported the differentiation of a very high percentage of human preadipocytes which developed a sensitive β-adrenergic lipolytic response but which lacked an α₂-adrenergic antilipolytic response.     

IGF-I- and IGFBP-3-expression in cultured human preadipocytes and adipocytes.

The expression and secretion of IGF-I and IGFBP-3 were investigated in cultured human preadipocytes and in in vitro differentiated adipocytes derived from human subcutaneous adipose tissue under chemically defined culture conditions. Human preadipocytes expressed mRNAs for IGF-I and IGFBP-3 and secreted the corresponding proteins into the culture medium as measured by sensitive radioimmunoassays. In human adipocytes; specific mRNA-expression was comparable to that found in preadipocytes, but IGF-I secretion was increased 10-fold (3.87 +/- 0.69 vs. 0.41 +/- 0.11 ng/ml/10(6) cells/48 hrs, p < 0.05) and IGFBP-3 secretion 2.5-fold (7.34+/-1.15 vs. 3.27+/-0.38 ng/ml/10(6) cells/48 hrs, p<0.05) in the presence of adipogenic medium probably resulting in an increase of unbound IGF-I. Under serum-free, chemically defined conditions human growth hormone (hGH) and insulin were found to be positive regulators and cortisol was found to be a negative regulator of IGF-I and IGFBP-3 secretion in preadipocytes. In cultured human adipocytes, hGH showed no effect on IGF-I and IGFBP-3 secretion, whereas insulin stimulated and cortisol inhibited the secretion of both proteins. We conclude that IGF-I and IGFBP-3 may not only exert their actions in human adipose tissue via circulation, but also in an auto/paracrine way.     

Changes in IGF-I receptor and IGF-I mRNA during differentiation of 3T3-L1 preadipocytes

Insulin-like growth factor-1 (IGF-I) is an essential factor for the differentiation of preadipocytes into adipocytes. We investigated the expression of IGF-I receptor and IGF-I RNA messenger during 3T3-L1 preadipocyte differentiation. Levels of IGF-I receptor decreased in the mature adipocytes compared to cells before the initiation of differentiation. In addition, cultures not induced to differentiate showed a decrease on the receptor levels after 4 days in the presence of insulin compared to cultures without treatment. The levels of the IGF-I RNA messenger were shown to be higher in mature adipocytes compared to preadipocytes. We propose an autocrine and/or paracrine action of IGF-I in this adipocyte differentiation model, where IGF-I produced by the differentiating preadipocytes acts over their adjacent cells and, in this way, diminishes the expression of IGF-I receptor.     

Transformation/neodifferentiation of human skin fibroblasts by acute oncornaviruses and dexamethasone

We demonstrated that the Kirsten murine sarcoma virus (KiMSV) and the Harvey murine sarcoma virus (HaMSV) converted human skin fibroblasts (HSF) into adipocytes. Adipocytic conversion of HSF by KiMSV and HaMSV was dependent on the presence of glucocorticosteroids. The Kirsten murine leukemia virus, the Harvey murine sarcoma [corrected] virus and the amphotropic helper virus (AP292) were ineffective by themselves. Balb murine sarcoma virus and Moloney murine sarcoma virus were, to a lesser degree, able to effect adipocytic conversion of HSF. In contrast, the feline sarcoma virus and the simian sarcoma virus did not cause this conversion. Together, the results suggest a role for certain oncogenes and glucocorticosteroids in the transformation/neodifferentiation of human cells.     

Quantitative Characteristics of the Transformation of Hamster Cells by PARA (Defective Simian Virus 40)-Adenovirus 7

An in vitro method for the quantitative measurement of transformation in hamster embryo fibroblasts by the PARA [defective simian virus 40 (SV40)]-adenovirus 7 hybrid has been developed. Transformation by PARA particles followed one-hit kinetics with a ratio of 1 focus-forming unit per 250 plaque-forming units. The method of viral adsorption had a direct effect upon the total number of foci which developed but not on the quantitative aspects of this assay. A fluorescent-focus assay was developed which provided a direct correlation of the observed morphological transformation and the presence of the PARA genome. This fluorescent-focus assay utilized detection of the SV40 tumor antigen, which was present in all foci transformed by PARA. Single foci induced by PARA were isolated and grown into cell lines. Two types of foci were observed and isolated; the first contained cells having a cuboidal or SV40-type morphology, and the second consisted of epithelial or adenovirus-type transformed cells. Both types contained the SV40 tumor and SV40 surface antigens as determined by the indirect fluorescence technique; however, only the epithelial cells contained the adenovirus 7 tumor antigen. All five cell lines which were injected into weanling Syrian hamsters were found to be oncogenic. These cell lines induced antibodies to both SV40 and adenovirus 7 tumor antigens in tumor-bearing animals.     

v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage- independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage- independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.     

Development of transformed phenotype induced by a human ras oncogene is inhibited by interferon

Mouse IFN inhibited the development of transformed foci in NIH 3T3 cultures transfected with the viral Ha-MuSV(ras) and Mo-MuSV(mos) oncogenes, or with the human bladder carcinoma ras EJ/T24 DNA. IFN treatment five or seven days after transfection was still effective in inhibiting the oncogenic transformation, but did not inhibit significantly the biochemical transformation induced by pSV2-neo or Ecogpt DNA, so that inhibition of ras-induced transformation was not a result of a general effect on the transfection process. Treatment with IFN did not alter the expression of ras EJ/T24 DNA after the transformed phenotype had been established.     

Expression of protein HC on the plasma membrane of different human cell types.

The surface expression of a recently described plasma glycoprotein called human complex-forming glycoprotein, hetergeneous in charge (protein HC) on a number of different human cell types was investigated. By means of direct and indirect immunofluorescence, protein HC was shown to be associated with the surface of virtually all cells of the investigated normal cell types including erythrocytes, peripheral blood B and T lymphocytes, and the human fibroblast lines HE 81, HE 31, and WI 38. When transformed and malignant cell populations were studied, it was found that some populations (e.g., the T cell line Molt-4) carried protein HC on the surfaces of very few cells, whereas other cell populations (e.g., chronic lymphocytic leukemia lymphocytes) carried the protein on most cells. Malignant cell populations with intermediary percentages of protein HC-positive cells were also found. Protein HC on the cell surface of normal peripheral blood lymphocytes could be redistributed by incubation of the cells with anti-protein HC-antiserum at 37 degrees C, and this reaction could be inhibited by sodium azide.     

In vitro hemopoiesis within a microenvironment created by MC3T3-G2/PA6 preadipocytes

The clonal preadipose cell line, MC3T3-G2/PA6, has the capacity to differentiate into adipocytes in response to glucocorticoids and to support in vitro growth of hemopoietic stem cells (CFU-S). To study the relationship between these capacities, we precultured the MC3T3-G2/PA6 cells for varying days in the presence or absence of dexamethasone and then cocultured them with mouse bone marrow cells. Logarithmically growing cultures contained no detectable adipocytes and showed the highest growth-supporting activity for CFU-S, whereas cultures containing the largest number of adipocytes showed the lowest activity. When bone marrow cells were seeded onto 3-day-old MC3T3-G2/PA6 preadipocyte layers at 1 X 10(5) cells/35-mm dish, day 12 CFU-S grew with a population doubling time of about 37 hr, and at least 75% of them were associated with the cell layer between days 2 and 7. In the absence of the preadipocytes, CFU-S were not detected in the adherent cell fraction and decreased with a half-life of about 18 hr. More than 80% of CFU-C were also found to be associated with the preadipocyte layer, and they increased about 24-fold in number during 7 days in culture. Morphologically, hemopoietic cells developing into mature granulocytes and macrophages were distributed between the layers of preadipocytes. Dendritic processes of preadipocytes were frequently in close alignment with the hemopoietic cells. However, adipocytes failed to show such an intimate association with hemopoietic cells. These results indicate that MC3T3-G2/PA6 cells in the preadipocyte stage, but not in the adipocyte stage, have the capacity to support CFU-S growth, and that hemopoiesis in our cocultivation system proceed within the microenvironmental milieu provided by MC3T3-G2/PA6 preadipocytes.     

Rodent cell transformation assays-a brief historical perspective

In vitro cell transformation is a process characterized by a series of progressive distinctive events that often emulate manifestations occurring in vivo and which are associated with neoplasia. Attendant cellular and sub-cellular alterations include, among others: cellular immortality, phenotypic changes, aneuploidy, genetic variability, cellular disarray, anchorage-independent growth, and tumorigenicity in vivo. Early chemically induced neoplastic transformation studies involved the use of normal diploid (Syrian) hamster embryo (SHE) cells and monitored the formation of morphologically altered colonies. Later investigations employed primarily two established mouse cell lines, i.e. the BALB/c 3T3 A31 cell line and the C3H 10T 1/2 cell line, and monitored the induction of morphologically aberrant foci. In either case, such transformed cellular clusters (colonies and foci) could induce tumors upon inoculation in vivo. Some subsequent noteworthy advancements using these systems included pH adjustments, metabolic supplementation, amplification of expression of formerly latent transformed foci, concurrent detection of mutagenesis and transformation, and use of a Bhas 42 cell line (v-Ha-ras transfected BALB/c 3T3 cells) to detect both tumor initiators and promoters. Over time, such transformation assay systems have been found useful in academic, industry and regulatory laboratories, generally for research purposes, but also occasionally as screening tools for potential chemical carcinogens. Nevertheless, to date, use of these assays for decision-making purposes in the regulatory arena remains elusive and will require comprehensive validation to gain universal acceptance.