p21Waf1 is required for complete oncogenic transformation of mouse embryo fibroblasts by E1Aad5 and c-Ha-ras oncogenes

Cyclin-dependent kinase inhibitor p21^Waf1 is known to have alternative functions associated with positive regulation of proliferation, actin cytoskeleton remodeling and suppression of apoptosis. The goal of the present study was to assess the role of p21^Waf1 in the establishment of the transformed phenotype of mouse embryo fibroblasts with stable expression of E1Aad5 and c-Ha-ras complementary oncogenes. Herein, we demonstrate that E1A/c-Ha-Ras-transformed p21^Waf1-null fibroblasts possess some characteristic features of transformed cells, such as loss of contact inhibition, high saturation density, shortened cell cycle, inability to undergo cell-cycle arrest after DNA damage and serum deprivation, but, at the same time, they are not completely transformed in that they are unable to proliferate at clonal density, are anchorage-dependent, retain a fibroblast-like morphology with pronounced actin cytoskeleton and show reduced migration and invasion. Our data support the concept of p21^Waf1 “tumor suppressor” having alternative oncogenic functions in the cytoplasm and for the first time indicate that p21^Waf1 can be indispensable for complete oncogenic transformation.     

The Ha-ras-induced transformed phenotype of rat-1 cells can be suppressed in hybrids with rat embryonic fibroblasts

Somatic cell hybrids were isolated from fusions of diploid embryonic rat fibroblasts with transformed Rat-1 cells which contained 4 to 5 copies of the transforming human Ha-ras 1 gene. In contrast to their transformed parental cells four hybrid clones showed normal morphology, long latency periods of tumorigenicity in newborn rats, anchorage requirement of proliferation, and an eightfold-reduced amount of secreted transforming growth factor activity. Thus these hybrids are called suppressed with regard to expression of the Ha-ras-induced transformed phenotype. Tumorigenic derivatives of the suppressed hybrids that had segregated chromosomes were isolated. Since two of the tumorigenic hybrid clones showed the similar low level of secreted transforming growth factors as the suppressed hybrids, decreased production of transforming growth factor activity is unlikely to be a sufficient criterion for suppression of malignancy. Whereas one of the suppressed hybrids expressed the transforming gene product p21 at a level similar to that of the transformed parental cells, other suppressed hybrids expressed less p21. This suggests that the suppressed phenotype can be regulated at the posttranslational level of p21 but that additional controls of expression of p21 are likely to exist. DNA of the suppressed hybrids transformed Rat-1 cells to proliferation in the presence of semisolid agar. Thus the activated human Ha-ras gene in the suppressed hybrids retained its biological activity even though it did not transform these cells to tumorigenicity.     

Increased levels of mitochondrial gene expression in rat fibroblast cells immortalized or transformed by viral and cellular oncogenes.

Steady-state levels of the mitochondrial (mt) mRNA encoding subunit II of cytochrome oxidase (COII) were increased 5-10 fold in fully transformed cell lines derived from rodent embryonic fibroblasts after transfer of polyoma virus DNA, and in immortalized cell lines established by transfer of plt (polyoma large T protein), E1A (adenovirus) and myc oncogenes. Increased mitochondrial gene expression was not related with active growth per se: it was low in fast-growing rat embryo cells, and it did not change upon serum starvation and subsequent stimulation of FR3T3 cells. The number of copies of mtDNA did not vary, and different mitochondrial mRNAs and rRNAs were increased in the same proportions, suggesting a change in the rate of accumulation of their common precursor.     

v-myb transformation of Xeroderma pigmentosum human fibroblasts: overexpression of the c-Ha-ras oncogene in the transformed cells

Human Xeroderma pigmentosum "normal" fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental "normal" AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.     

The c-fos proto-oncogene promotor is not regulated by serum, epidermal growth factor, and phorbol ester in embryonal fibroblasts transformed by E1Aad5+cHa-ras-oncogenes]

Regulation of c-fos protooncogene activity in rat embryonal fibroblasts (REF), E1Aad5-immortalized REF cells, and E1Aad5 + cHa-ras transformed REF cells has been investigated. The analysis of regulation of fos-promoter activity was done by means of transient and stable transfection of fos-CAT plasmid into immortalized and transformed cells. In parallel, the regulation of cellular c-fos as well as c-jun and c-myc genes expression has been studied. It has been found that in E1Aad5 + cHa-ras-transformed cells the expression of c-fos promoter has a constitutive, non-inducible character while in REF cells and cells immortalized by E1Aad5 the fos-promoter can be regulated by serum growth factors, EGF, and TPA.     

Apoptotic cell death in rat embryo fibroblasts transformed by EiA + cHa-RAS oncogenes after gamma irradiation

A mechanism of apoptotic death of normal rat embryo fibroblasts and of those transformed by E1A + cHa-Ras oncogenes following gamma irradiation has been investigated. The E1A + cHa-Ras transformed cells were shown to express wild type p53 which was able to trans-activate a reporter pG13-luc Plasmid. As a result of trans-activation, an accumulation of universal inhibitor of cyclin-dependent kinases--p21/Waf1 protein and an increase in the proportion of p21/Waf1 expressing cells were observed, The accumulated p21/Waf1 was found to bind with PCNA. The association with PCNA, however, did not lead to suppression of DNA replication according to the data of iododeoxyuridine (IdUr) incorporation. A high proportion of S-phase cells, in combination with cell cycle blocking in G2-phase, promoted polyploidization of E1A + cHa-Ras transformed cells after gamma irradiation. The polyploidic cells with DNA content equal and higher than 8c die 48-72 h following irradiation due to apoptosis. A significant proportion of E1A + cHa-Ras cells with incorporated IdUr contains labeled micronuclei, the fact being a morphological evidence of apoptosis of cells in S-phase of the cell cycle.     

Function of the adenovirus E1B oncogene in infected and transformed cells

Adenovirus infection and E1A gene expression stimulates cellular proliferation as a mechanism to facilitate virus replication. Programmed cell death (apoptosis) is the cellular response to this deregulation of growth control by E1A during viral infection and neoplastic transformation. To combat the suicidal elimination of virus infected cells by apoptosis, adenovirus has evolved a mechanism to disengage the apoptotic program of the cell. This anti-apoptotic function is encoded within the adenovirus E1B 19 kDa and 55 kDa gene products. Both viral products encoded by E1B act at independent and overlapping points in the cell death process to ensure that the premature death of the host cell does not take place and that viral infection can progress to completion. The E1B 55K protein functions as an anti-apoptotic gene product by direct physical interference with the p53 tumor suppressor protein, whereas the E1B 19K protein acts to inhibit p53-dependent and probably p53-independent apoptosis by a mechanism that resembles that of the human bcl-2 protooncogene.     

The characteristics of actin cytoskeleton structure and its rearrangements by extracellular matrix proteins in normal, immortalized and transformed rat fibroblasts

Comparative analysis of actin cytoskeleton structure in rat embryonic fibroblasts, E1A-immortalized and E1A + cHa-ras-transformed cells has been carried out. A decrease in adhesiveness and the rate of changes in actin cytoskeleton structures was shown to correlate with the level of morphological transformation of cells. E1A + cHa-ras-transformants show the lowest adhesiveness and complete disorganization of actin structures. Cultivation on serum-free media promoted disassembling of actin cytoskeleton structures in a small part of normal fibroblast population, only in a few immortalized cells, but exerted no influence on transformed cells. The influence of immobilized extracellular matrix proteins fibronectin, laminin and collagens type I and III on actin cytoskeleton structure in normal, immortalized and transformed fibroblasts was studied. Transformed cells spread on fibronectin completely restored highly organized actin structures, displayed a lot of stress fibers and focal contacts. The use of laminin revealed differences in locomotion between normal and transformed cells. Normal, immortalized and transformed fibroblasts spread on fibronectin and laminin demonstrate some peculiarities in actin cytoskeleton structures as a result of specificity of ligand-receptor interaction. Cells spread on fibronectin have polygonal shapes, many stress fibers and focal contacts, whereas cells spread on laminin are highly polarized and develop broad lamellae filled with actin microfilament meshwork. Collagens type I and III can affect adhesive properties and actin cytoskeleton structure in all cell lines studied only slightly, in comparison with fibronectin and laminin.     

Rat embryo fibroblasts transformed by complementation with oncogenes E1A+E1B-19 and E1A+cHa-ras differ in the ability to realize the G1/S block in serum free media

The capability of REF cells transformed by EA + E1B-19 kDa and EA + cHa-ras oncogenes to realize the G1/S cell cycle arrest upon serum starvation was studied. The amount of cyclin-kinase inhibitor protein p27/Kip was shown to increase in both normal and transformed cells. However, the p27/Kip-bound cyclin-kinase complexes of transformed cells were found to be active, implying the functional inactivation of p27/Kip inhibitor. Nevertheless, in contrast to E1A + cHa-ras transformants, E1A + E1B-19 kDa transformants undergo the G1 cell cycle arrest. The G1 cell cycle block correlates with the decrease in cyclinE-Cdk2 activity. Since cyclinE-Cdk2 complexes need Thr-160 phosphorylation of Cdk2 by CAK-kinase for full activity, we have analysed the Cdk-7 associated activity upon serum starvation using gst-Cdk2 as a substrate. Serum starvation did not affect CAK activity either in E1A + cHa-ras or in E1A + E1B-19 kDa transformants. Thus, selective suppression of cyclineE-Cdk2 activity in E1A + E1B-19 kDa transformants upon serum starvation does not arise from the action of cyclin-kinase inhibitors, or from change in CAK activity.     

The regulation of protein kinase C activity in normal, immortalized and transformed rat fibroblasts]

In view of recent studies showing that cell proliferation of E1Aad5+c-Ha-ras-transformed fibroblasts cannot be regulated by growth factors and phorbol eaters in contrast to normal and E1Aad5-immortalized cell lines, the present work was undertaken to examine the role of protein kinase C (PKC) in the mitogenic signal transduction machinery in rat embryonal fibroblasts. It is shown that PKC is activated by acidic growth factor and phorbol esters in all the three cell lines. These findings suggest the existence of an additional, not associated with PKC-, growth-signaling pathway in E1Aad5-Ha-ras-transformed rat embryonal fibroblasts.     

Differential induction of cytolytic susceptibility by E1A, myc, and ras oncogenes in immortalized cells.

The E1A oncogene of adenovirus serotypes 2 and 5 induces susceptibility to the cytolytic effects of natural killer lymphocytes and activated macrophages when expressed in infected and transformed mammalian cells (cytolysis-susceptible phenotype). E1A and the oncogenes v-myc, long-terminal-repeat-promoted c-myc, and activated c-ras share the ability to immortalize transfected low-passage rodent cells. The cytolytic phenotypes of well-characterized rodent cell lines immortalized by these three oncogenes were defined. In contrast to target cells expressing the intact E1A gene, myc- and ras-expressing, immortalized primary transfectants were resistant to lysis by both types of killer cell populations. The same patterns of susceptibility (E1A) and resistance (myc and ras) to cytolysis were observed in oncogene-transfected continuous rat (REF52) and mouse (NIH 3T3) cell lines, indicating that differences in the cytolytic phenotypes associated with expression of these oncogenes are not due to cell selection during immortalization. The results suggest that the E1A oncogene may possess a functional domain that is different from those of other oncogenes, such as myc and ras, and that the activity linked to this postulated domain is dissociable from the process of immortalization.     

Histone deacetylase inhibitor blocks proliferation of cells transformed with oncogenes E1A and cHa-ras

Rat embryonic fibroblasts, transformed with E1A and cHa-ras oncogenes, are unable to stop in the cell cycle checkpoints under growth factor withdrawal and genotoxic stresses (Bulavin et al., 1999). In the present paper, we showed that sodium butyrate, an inhibitor of histone deacetyase activity, decreased the share of cells being in S-phase, and caused G1/S and G2/M blocks of the cell cycle in the transformants. By means of RT-PCR and immunoblotting, we found that NaB significantly changed the expression of genes involved in proliferation: cyclins D1, A, E and cyclin-dependent kinases Cdk2 and Cdk4, whereas the amount of p21Waf1 and p27Kip1 inhibitors greatly increased. Along with accumulation of p21Waf1 protein content, that of Cdk2-bound p21 increases. Taken together, these data allow to suggest that NaB treatment does evidently restore the capability of p21Waf1 to inhibit cyclin-kinase activity. One may suppose that inhibition of HDAC activity by sodium butyrate leads to activation of yet unknown HDAC-dependent genes, which is followed by restoration of p21Waf1 function in spite of the E1A oncogene expression.     

Morphological transformation and tumorigenicity in C3H/10T1/2 cells transformed with an inducible c-Ha-ras oncogene

A C3H/10T1/2 cell line containing an inducible metallothionein-ras hybrid oncogene was conditionally and reversibly transformed upon exposure to zinc ions. Interestingly, although the cell line was fully malignant when expressing only low levels ofras, complete morphological transformation required much higher levels.     

Expression of interstitial collagenase and its endogenous regulators in immortalized and transformed by HPV-16 E7 gene fibroblasts

Matrix metalloproteinases (MMPs) play a critical role in tumor development and invasion. The aim of this study was to elucidate peculiarity of expression of interstitial collagenase (MMP-1) and its endogenous regulators during oncogenic transformation of fibroblasts by HPV-16 E7 gene. Papilloma virus types 16 and 18 are etiological factor of cervical cancer. We have studied expression of MT1-MMP, MMP-1, tissue inhibitor of these proteases, TIMP-1, and urokinase-like plasminogen activator (uPA), activating MMP-1 via plasmin. The study was carried out using fibroblasts immortalized by LT gene (IF) and transformed by E7 gene of HPV-16 fibroblasts (TF). Primary culture of Fisher rat embryo fibroblasts was used as a control (PF). mRNA expression, and enzymatic activity were studied by RT-PCR and by hydrolysis of fluorogenic type I collagen, respectively. Cell transformation was accompanied by: (a) 2–3 fold induction of MT1-MMP mRNA expression vs PF; (b) the decrease in mRNA level of TIMP-1 (1,5–2 fold); c) unchanged uPA expression. Cell immortalization is accompanied by: (a) the increase of MT1-MMP expression (1,5–2 fold); (b) unchanged TIMP-1 expression; (c) the increase of uPA expression (2–4 fold) vs PF and TF. MMP secreted activity and activity in lysates of TF increased but level of free endogenous MMP inhibitors decreased vs IF. Data on gene expression are consistent with enzymatic data on the collagenolytic activity. These results suggest changes in enzyme/inhibitor/activator ratio both TF and IF and significant enhancement of the destructive potential of the TF.