Search PubMed⌕ Search

Biomedical subjects

C Cerni

Publications and source records attributed to C Cerni.

At least 19 recordsLinked to original sources
PubMed2000

Telomeres, telomerase, and myc. An update.

Normal human somatic cells have a finite life span in vivo as well as in vitro and retire into senescence after a predictable time. Cellular senescence is triggered by the activation of two interdependent mechanisms. One induces irreversible cell cycle exit involving activation of two tumorsuppressor genes, p53 and pRb, and the proper time point is indicated by a critical shortening of chromosomal ends due to the end-replication problem of DNA synthesis. The development of a malignant cancer cell is only possible when both mechanisms are circumvented. The majority of human cancers and tumor cell lines produce telomerase, a ribonucleoprotein with two components required for core enzyme activity: telomerase RNA (TR) and a telomerase reverse transcriptase protein (TERT). Telomerase adds hexameric DNA repeats (TTAGGG) to telomeric ends and thus compensates the progressive loss of telomeric sequences inherent to DNA replication. While TR of telomerase is present in almost all human cells, human TERT (hTERT) was found rate limiting for telomerase activity. Ectopic expression of hTERT in otherwise mortal human cells induced efficient elongation of telomeres and permanent cell growth. While hTERT-mediated immortalization seems to have no effect on growth potential and cell cycle check points, it bestows an increased susceptibility to experimental transformation. One oncogene that might activate TERT in the natural context is c-myc. Myc genes are frequently deregulated in human tumors and myc overexpression may cause telomerase reactivation and telomere stabilization which, in turn, would allow permanent proliferation. Is this a general strategy of incipient cancer cells to escape senescence? Several recent observations indicate that other scenarios may be conceived as well.

Animals↗
PubMed1999

Modulation of invasive properties of murine squamous carcinoma cells by heterologous expression of cathepsin B and cystatin C.

Murine SCC-VII squamous carcinoma cells have the capacity to penetrate reconstituted basement membranes (Matrigel) in vitro. The invasion of Matrigel layers by SCC-VII cells was significantly reduced by E-64, a specific inhibitor of lysosomal cysteine proteinases. The cathepsin-B-selective E-64 derivative, CA-074, inhibited penetration of Matrigel by SCC-VII cells to the same extent, indicating a major role for this particular lysosomal enzyme in extracellular-matrix degradation during squamous-carcinoma-cell invasion. SCC-VII cells were stably transfected with a cDNA encoding human procathepsin B, in an attempt to modulate the invasive properties of the cell line. The transfected cells expressed the heterologous gene, secreted increased amounts of procathepsin B and displayed enhanced invasive potential. In vivo, the activity of cathepsin B is strictly regulated by endogenous inhibitors. SCC-VII cells were therefore also stably transfected with a cDNA encoding human cystatin C, the most potent cysteine-proteinase inhibitor in mammalian tissues. The expression of this transgene resulted in the production of active recombinant cystatin C and a pronounced reduction in Matrigel invasion. These studies demonstrate that the invasive properties of squamous-cell carcinomas can be changed by modulation of the balance between cathepsin B and its endogenous inhibitors, and provide further evidence for the involvement of this lysosomal cysteine proteinase in tumour invasion and metastasis.

Animals↗
PubMed1998

YY1 can inhibit c-Myc function through a mechanism requiring DNA binding of YY1 but neither its transactivation domain nor direct interaction with c-Myc.

The proto-oncoprotein c-Myc and the multifunctional transcriptional regulator YY1 have been shown previously to interact directly in a manner that excludes Max from the complex (Shrivastava et al., 1993). As binding to Max is necessary for all known c-Myc activities we have analysed the influence of YY1 on c-Myc function. We demonstrate that YY1 is a potent inhibitor of c-Myc transforming activity. The region in YY1 required for inhibition corresponds to a functional DNA-binding domain and is distinct from the domains necessary for direct binding to c-Myc. Furthermore the transactivation domain of YY1 was not necessary suggesting that gene regulation by YY1, for example through DNA bending or displacement of regulators from DNA, could be the cause for the negative regulation of c-Myc. This model of indirect regulation of c-Myc by YY1 was supported by the finding that although YY1 did not bind to the c-Myc transactivation domain (TAD) in vitro it was able to inhibit transactivation by Gal4-MycTAD fusion proteins in transient transfections. As for the inhibition of transformation, an intact DNA-binding domain of YY1 was necessary and sufficient for this effect. In addition YY1 did not alter c-Myc/Max DNA binding, further supporting an indirect mode of action. Our findings point to a role of YY1 as a negative regulator of cell growth with a possible involvement in tumor suppression.

Alanine↗
PubMed1997

Expression of mouse RNase MRP RNA in human embryonic kidney 293 cells.

We report on the expression of mouse RNase MRP RNA in human embryonic kidney 293 cells upon DNA transfection. Stable cell lines were selected by cotransfection with a neor gene. Transcription of wild-type and deletion mutants of MRP RNA and ribonucleoprotein formation were assessed by RNase protection and immunoprecipitation experiments. Mouse MRP RNA as expressed in 293 cells readily associates with human proteins to form a chimeric Th ribonucleoprotein. 5' truncated MRP RNAs, however, failed to associate with Th antigen(s) and deletion of the 3' sequences of MRP RNA greatly reduced the expression in stable as well as in transient transfectants.

Animals↗
PubMed1997

Oxidized low-density-lipoprotein triggers programmed death of endothelial cells.

Incubation of bovine aortic as well as human umbilical vein endothelial cells with either oxidized or native low-density-lipoprotein in the presence of trace amounts of copper induced morphological changes of the cells and chromatin fragmentation characteristic for programmed cell death. Shrinkage of cells was evident after 6 to 8 hours of incubation and clearly preceded release of lactate dehydrogenase as a marker of cell permeability. Condensation of nuclear chromatin and internucleosomal cleavage was demonstrated by Hoechst staining and gel electrophoresis, respectively. Thus, by inducing active death of endothelial cells oxidized low-density-lipoprotein might negatively influence tissue homeostasis of the endothelium and thereby promote the development of atherosclerotic plaques.

Animals↗
PubMed1996

ADP-ribosylation of wild-type p53 in vitro: binding of p53 protein to specific p53 consensus sequence prevents its modification.

We have recently reported that mutant but not wild-type (wt) p53 protein was ADP-ribosylated in primary rat cells overexpressing the temperature-sensitive murine p53val135 gene. To examine whether the lack of susceptibility to modification is a specific feature of p53val135 adopting wt conformation or rather a general property of this tumor suppressor protein, we have studied ADP-ribosylation of wt p53 of different origin in vitro using semi-purified poly(ADP-ribose) transferase (pADPRT). In vitro pADPRT modified human and mouse wt p53 and p53val135. Under limiting substrate concentration, the molar mass of ADP-ribosylated p53 was only slightly altered. Chase experiments with high NAD concentration resulted in the formation of poly(ADP-ribosyl)ated p53 protein shifted to 64 kD. However, preincubation of wt p53 proteins with a p53 consensus sequence resulting in complex formation abolished the modification of wt p53. This indicates that in the cellular environment the specific DNA binding of wt p53 prevents its covalent modification by poly(ADP-ribose).

Adenosine Diphosphate Ribose↗
PubMed1996

ADP-ribosylation of p53 tumor suppressor protein: mutant but not wild-type p53 is modified.

Poly(ADP-ribosyl)ation of mutant and wild-type p53 was studied in transformed and nontransformed rat cell lines constitutively expressing the temperature-sensitive p53135val. It was found that in both cell types at 37.5 degrees C, where overexpressed p53 exhibits mutant conformation and cytoplasmic localization, a considerable part of the protein was poly(ADP-ribosyl)ated. Using densitometric scanning, the molecular mass of the modified protein was estimated as 64 kD. Immunofluorescence studies with affinity purified anti-poly(ADP-ribose) transferase (pADPRT) antibodies revealed that, contrary to predictions, the active enzyme was located in the cytoplasm, while in nuclei chromatin was depleted of pADPRT. A distinct intracellular localization and action of pADPRT was found in the cell lines cultivated at 32.5 degrees C, where p53 adopts wild-type form. Despite nuclear coexistence of both proteins no significant modification of p53 was found. Since the strikingly shared compartmentalization of p53 and pADPRT was indicative of possible complex formation between the two proteins, reciprocal immunoprecipitation and immunoblotting were performed with anti-p53 and anti-pADPRT antibodies. A poly(ADP-ribosyl)ated protein of 116 kD constantly precipitated at stringent conditions was identified as the automodified enzyme. It is concluded that mutant cytoplasmic p53 is tightly complexed to pADPRT and becomes modified. At 32.5 degrees C binding to DNA of p53 or its temperature-dependent conformational alteration might prevent an analogous modification of the tumor suppressor protein.

Adenosine Diphosphate Ribose↗
PubMed1995

Differential effects by Mad and Max on transformation by cellular and viral oncoproteins.

c-Myc is an essential component of the regulatory mechanisms controlling cell growth. Max is the obligatory partner of c-Myc for all its biological functions analysed to date. Recently two Max interacting proteins, Mad and Mxi1, have been identified. It has been suggested that these two proteins modulate c-Myc function, in the simplest model by competing with c-Myc for the interaction with Max. We have analysed different aspects of Mad function in comparison to Max. Native Mad/Max heterodimers bound specifically to a c-Myc/Max consensus DNA binding site. Furthermore Mad inhibited efficiently c-Myc, mutant p53, adenovirus E1a, or human papilloma virus type 16 transformation of rat embryo cells in cooperation with activated Ha-Ras. Myc transformed clones showed an increased cell cycle time and a reduced immortalization frequency after cotransfection with either mad or max. In contrast to Mad, Max did not inhibit E1a/Ha-Ras cotransformation but repressed c-Myc/Ha-Ras transformation efficiently. Mad delta N, an N-terminal deletion mutant of Mad, was as efficient in repressing c-Myc/Ha-Ras cotransformation as full length Mad but showed little inhibitory activity when assayed on E1a/Ha-Ras. Unlike wt Mad, Mad delta N had little effect on cell growth. Our data suggest that Mad affects cell growth at least in part by a c-Myc independent mechanism.

Animals↗
PubMed1991

Immunization against human papillomavirus type 16 tumor cells with recombinant vaccinia viruses expressing E6 and E7.

Papillomaviruses are etiological agents of epithelial proliferative disease. In man, neoplastic transformation of the uterine cervix has been linked to infection with specific subtypes of human papillomavirus, particularly types 16 and 18. We previously reported that live vaccinia virus recombinants expressing early transforming proteins of other tumor viruses can immunize against challenge with cognate tumor cells and we have extended this approach to HPV16. Neoplastic transformation by papillomaviruses involves expression of early open reading frames (ORFs) E5, E6, and E7, and we report the construction of vaccinia recombinants separately expressing ORFs E5-E7 of HPV16. Primary rat cell lines cotransformed with HPV16 and an activated ras oncogene were established in order to evaluate the potential of the recombinants to elicit antitumor immunity. We report that inoculation of rats with vaccinia recombinants expressing E6 or E7 retarded or prevented tumor development in a proportion of animals challenged by subcutaneous seeding of tumor cells whereas the recombinant expressing E5 was inactive.

Animals↗
PubMed1990

Immortalization of primary rat embryo cells by human papillomavirus type 11 DNA is enhanced upon cotransfer of ras.

Transfer of human papillomavirus type 11 (HPV11) DNA and a neo(r) marker into primary rat embryo cells (REC) led to colony formation in G418-selective medium. About 20% of HPV11 clones were eventually established in culture but displayed low growth rates. Cotransfection of HPV11 DNA and an activated ras oncogene led to formation of both drug-resistant flat colonies and phenotypically transformed clones which grew efficiently when expanded in culture. A number of transformants reverted to a flat, "normal" morphology shortly after isolation. Nontransformed clones expressed only HPV11 genes, while those maintaining a transformed phenotype transcribed both ras and HPV11 genes efficiently and were highly tumorigenic. Expression of HPV11 thus seems, necessary for induction of colony formation, but efficient long-term growth seems to require at least the transient presence of ras.

Animals↗
PubMed1990

Non-selective analysis of the transformation of FR3T3 rat cells by bovine papillomavirus type 1: regulations of viral transcription associated with phenotypic transformation.

Drug-resistant clones selected from FR3T3 rat cells after transfer of neo-BPV1 (Bovine Papillomavirus Type 1) DNA constructs became phenotypically transformed (focal transformation, growth in suspension and tumor formation) soon after selection (approximately 5 generations in culture). A frameshift mutation in ORF E5 abolished transformation, but did not prevent the autonomous maintenance of the DNA construct. A more complex situation was observed when the E2 transactivating function was abrogated. A minority of the E2(-)-neor clones became phenotypically transformed shortly after drug selection, but the majority maintained normal growth properties for 30 to 50 generations. The rate of viral transcription was uniformly high in cells which exhibited transformed growth properties early after selection (the E2- minority class and all the wild type transformants) and low in phenotypically normal cells (the majority of the E2- lines). The same low transcriptional activity and delayed expression of transformed growth properties had been observed after transfection of a similar construct carrying a wild type viral early region (69-T fragment), but lacking the late region. The elevated rate of viral transcription, which correlates with the immediate expression of transformation, appears therefore to require at least two distinct elements, the E2 transactivator function and sequences in the late region of the viral genome. In their absence, high transcription rates and transformation could be established only in a minority of the transfected clones, by an unknown, E2-independent mechanism. Evidence was obtained for a third transformation route which, in the absence of either E2 or the late region, led to the focal occurrence of transformed derivatives after 30 to 50 generations of normal growth, but was not associated with an overall increase in viral expression.

Animals↗
PubMed1989

Successive steps in the process of immortalization identified by transfer of separate bovine papillomavirus genes into rat fibroblasts.

Transfer of neor and bovine papillomavirus type 1 (BPV1) DNA into rat embryo fibroblasts led to colony formation in G418-containing medium, with no detectable background in controls with neor DNA alone. More than 50% of the drug-resistant clones could be further propagated in culture. The genetic functions of BPV1 involved in colony formation and in long-term immortalization were investigated by both translation termination mutations in the full-length genome, which inactivate individual open reading frames, and constructs in which these open reading frames were separately expressed under control of long terminal repeat promoter enhancers. Expression of either open reading frame E2 or E5 was sufficient for formation of a drug-resistant colony, but long-term growth in culture required that of E6. No significant cooperative effect was observed upon cotransfection of BPV1 and ras oncogene DNAs. Expression of the early region of the human papillomavirus type 16 also led to immortalization of rat embryo fibroblast cells in the same assay, and, unlike what was previously reported in baby rat kidney cells, it required neither activation by a heterologous promoter, nor a cooperating ras oncogene.

Animals↗
PubMed1988

Tumorigenic transformation of rat FR3T3 fibroblasts carrying an activated myc oncogene requires subsequent mutational events.

Early passage FR3T3 rat cells were co-transfected with a neo resistance gene and pSVc-myc-1, an SV40-driven expression vector carrying an activated murine myc oncogene. Selection in G418 medium produced clones expressing the exogenous myc gene at various levels, with a concomitant loss of expression of the normal c-myc allele. These clones were phenotypically normal, but, in fluctuation tests performed according to Luria and Delbrück (1943) on subcultures independently derived from the same clone, transformed foci appeared as stochastic events with a wide range of fluctuation. These results indicate that expression of the oncogene was not sufficient to induce the appearance of transformed growth properties, and that secondary genetic changes are required, most likely mutations in cellular proto-oncogenes. Within a single clone, independent transformants exhibited different tumorigenic potentials, spanning from high efficiency to no detectable tumor induction, without any clear correlation with their degree of in vitro transformation. Tumors and cell lines established from independent tumors, while maintaining the exogenous myc gene without gross rearrangement of its structure, no longer expressed the oncogene and resumed the expression of the normal allele.

Animals↗
PubMed1987

Transfer of 'immortalizing' oncogenes into rat fibroblasts induces both high rates of sister chromatid exchange and appearance of abnormal karyotypes.

Cell lines established after transfer into FR3T3 rat fibroblast cells of 'immortalizing' oncogenes (plt gene (large T protein) of polyoma virus, v-myc gene of MC29 virus, rearranged forms of c-myc) exhibited increased rates of sister chromatid exchange (SCE). This was observed neither in cells which expressed one of the oncogenes responsible for the terminal stages of tumorigenic transformation (polyoma virus pmt (middle T protein), mutated ras genes), nor in cell lines carrying oncogenes of both types. Abnormal chromosome numbers were observed in cell lines expressing plt or myc, but not after transformation by pmt or ras oncogenes.

Animals↗
PubMed1987

Elevated expression of an exogenous c-myc gene is insufficient for transformation and tumorigenic conversion of established fibroblasts.

Two established rat fibroblast lines, differing only by their number of generations in culture, show dramatically different responses to the elevated c-myc expression delivered by an efficient murine c-myc retrovirus vector. Thus, a late passage (60 generation) FR3T3 line acquires a transformed and tumorigenic phenotype upon introduction of this activated c-myc gene as indicated by its altered morphology, high efficiency of focus formation, soft agar clonability, saturation density in monolayer culture, and short latency of tumorigenicity in syngeneic hosts. Remarkably, none of these characteristics, except for an increased refractility in monolayers and an epidermal growth factor (EGF)-dependent agar clonability, were observed in a variety of early passage (10 generation) FR3T3 c-myc clones. BALB/c A31 fibroblasts transfected with this c-myc retroviral vector behaved essentially the same as the FR3T3 early line except for their inability to grow in suspension in response to EGF. However, transformation and tumorigenic conversion of each of these three fibroblast lines was achieved by an activated ras oncogene. Hence, elevated c-myc expression is insufficient for transformation of established fibroblasts but depends upon other acquired cooperating functions which are not necessary for ras induced transformation. We also demonstrate that endogenous c-myc expression remains unaffected even in clones expressing a 100-fold excess of exogenous c-myc RNAs demonstrating that c-myc autoregulation is not operative in these cells.

Animals↗