Christa Cerni

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.

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.

PARP-10, a novel Myc-interacting protein with poly(ADP-ribose) polymerase activity, inhibits transformation

The proto-oncoprotein c-Myc functions as a transcriptional regulator that controls different aspects of cell behavior, including proliferation, differentiation, and apoptosis. In addition, Myc proteins have the potential to transform cells and are deregulated in the majority of human cancers. Several Myc-interacting factors have been described that mediate part of Mycs functions in the control of cell behavior. Here, we describe the isolation of a novel 150 kDa protein, designated PARP-10, that interacts with Myc. PARP-10 possesses domains with homology to RNA recognition motifs and to poly(ADP-ribose) polymerases (PARP). Molecular modeling and biochemical analysis define a PARP domain that is capable of ADP-ribosylating PARP-10 itself and core histones, but neither Myc nor Max. PARP-10 is localized to the nuclear and cytoplasmic compartments that is controlled at least in part by a Leu-rich nuclear export sequence (NES). Functionally, PARP-10 inhibits c-Myc- and E1A-mediated cotransformation of rat embryo fibroblasts, a function that is independent of PARP activity but that depends on a functional NES. Together, our findings define a novel PARP enzyme involved in the control of cell proliferation.

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. Int. J. Cancer. 83:526–531, 1999.

Inhibitor of apoptosis protein (IAP) survivin is upregulated by oncogenic c-H-Ras.

Among the inhibitors of apoptosis proteins (IAPs), survivin has attracted special attention through its involvement in human cancer. The mechanism underlying tumour-associated survivin re-expression is not known. We found a correlation between exogenous c-H-Ras oncoprotein and endogenous survivin in a series of rat cell lines, which expressed defined oncogenes. Moreover, human HaCat cells, transfected with a constitutively activated c-H-ras gene, had significantly increased survivin levels. To study the interdependence of the two proteins, we generated a rat cell line that expressed a dexamethasone-inducible c-H-ras construct. Induction of c-H-Ras expression was followed by rapid upregulation of survivin. Conversely, downregulation of the oncoprotein resulted in prompt reduction of survivin to baseline value. c-H-Ras-induced survivin was expressed constitutively and independent of cell cycle progression or proliferation. Compromising Ras-stimulated PI3-K activity and MEK1 by chemicals abolished survivin expression and was associated with apoptotic cell death. Upregulation of survivin appeared to be an important activity of c-H-Ras oncoprotein, since cotransfection of a survivin-antisense construct into c-myc/c-H-ras-transfected primary rat embryo cells resulted in profound reduction of transformed clones. It is tempted to speculate that the frequent presence of survivin in human cancer cells might be a consequence of activated Ras-signalling pathways.

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°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 37.5°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 tighly complexed to pADPRT and becomes modified. At 32.5°C binding to DNA of p53 or its temperature‐dependent conformational alteration might prevent an analogous modification of the tumor suppressor protein.

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 (). 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.

The Human Trithorax Protein hASH2 Functions as an Oncoprotein

Regulation of chromatin is an important aspect of controlling promoter activity and gene expression. Posttranslational modifications of core histones allow proteins associated with gene transcription to access chromatin. Closely associated with promoters of actively transcribed genes, trimethylation of histone H3 at lysine 4 (H3K4me3) is a core histone mark set by several protein complexes. Some of these protein complexes contain the trithorax protein ASH2 combined with the MLL oncoproteins. We identified human ASH2 in a complex with the oncoprotein MYC. This finding, together with the observation that hASH2 interacts with MLL, led us to test whether hASH2 itself is involved in transformation. We observed that hASH2 cooperates with Ha-RAS to transform primary rat embryo fibroblasts (REF). Furthermore, transformation of REFs by MYC and Ha-RAS required the presence of rAsh2. In an animal model, the hASH2/Ha-RAS-transformed REFs formed rapidly growing tumors characteristic of fibrosarcomas that, compared with tumors derived from MYC/Ha-RAS transformed cells, were poorly differentiated. This finding suggests that ASH2 functions as an oncoprotein. Although hASH2 expression at the mRNA level was generally not deregulated, hASH2 protein expression was increased in most human tumors and tumor cell lines. In addition, knockdown of hASH2 inhibited tumor cell proliferation. Taken together, these observations define hASH2 as a novel oncoprotein.

Repression of in vivo growth of Myc/Ras transformed tumor cells by Mad1.

The Myc/Max/Mad network of transcriptional regulatory proteins plays an essential role in cell proliferation, growth, apoptosis, and differentiation. Whereas Myc proteins affect cell cycle progression positively, Mad proteins are negative regulators of cell proliferation. It has been shown in several in vitro systems that Mad proteins antagonize c-Myc functions. In this report we describe the inhibition of tumor cell outgrowth in vivo by Mad1 expression. Transformed cell lines were generated by co-transfection of c-myc, c-H-ras, and a chimeric mad1ER construct into primary rat embryo cells (MRMad1ER cells). Activation of Mad1 by 4-Hydroxy-Tamoxifen (OHT) resulted in abrogation of telomerase activity, reduced cloning efficiency, and decreased proportion of cells in S phase. Injection of MRMad1ER cells into syngenic rats induced aggressively growing tumors after a short latency period. This tumor growth was inhibited by OHT-treatment of animals, with the extent of inhibition correlating with the amount of OHT injected. No effect of OHT on tumor growth was observed with similarly transformed Myc/Ras cell lines which did not express Mad1ER. These data demonstrate that Mad1 is able to suppress Myc/Ras-mediated transformation under in vivo conditions.

Nuclear Ras: Unexpected subcellular distribution of oncogenic forms

The Harvey‐ras gene encodes small guanine nucleotide binding proteins, mutant forms of which are associated with a number of human malignancies. Based on studies with truncated forms of the protein it is known that correct post‐translational processing of Ras is essential for cytoplasmic membrane localization and function. Surprisingly, immunofluorescence analysis provided evidence that in addition to its cytosolic localization, activated H‐RasVal 12 was also localized in the nuclei of transformed cells both in vitro and in vivo. Immunoblot analysis of nuclear fractions was consistent with results found by immunohistochemistry. Moreover, inhibition of protein farnesylation prevented the nuclear targeting of activated H‐RasVal 12 and NFκB. Alterations in subcellular distribution pattern and phosphorylation of the cell cycle inhibitor p27, which is involved in Ras driven tumor growth, coincided with nuclear localization of H‐RasVal 12. Proteins are often not functional until they are transported to their final destination. Indeed, Ras was found to complex with NTF2 a factor involved in nuclear protein import and export. Therefore it is suggested that NTF2 is the actual carrier for oncogenic Ras. In view of these observations the question arises whether the nuclear localization of H‐RasVal 12 in tumors is important in oncogenic activation or whether it is a response to apoptosis. J. Cell. Biochem. Suppl. 36: 1‐11, 2001.