Biomedical subjects
Publications and source records attributed to M Tommasino.
A total of 21 squamous-cell carcinoma of the conjunctiva (SCC) and 22 control subjects had conjunctival samples tested for human papillomavirus (HPV) types using PCR-based assays. Epidermodysplasia verruciformis HPV types were found in 86% of SCC cases and 36% of control subjects (Odds ratio=12.0), suggesting a role of HPVs in the aetiology of SCC.
AIMS: To investigate whether the expression of interferon (IFN)-inducible gene IFI16 is inversely related to proliferative activity in vivo, we compared immunohistochemical reactivity of IFI16 in a series of head and neck squamous cell carcinomas (HNSCCs) with their proliferation index and the cell cycle regulator pRb. As human papillomavirus (HPV) infection is manifested by changes in the function or expression level of host genes such as IFN-inducible genes, we also investigated the presence of HPV DNA to determine whether head and neck cancers associated with HPV DNA can be distinguished from tumours that are presumably transformed by other mechanisms. METHODS: Thirty-six HNSCCs were evaluated for IFI16, pRb and Ki67 expression by immunohistochemistry. The presence of HPV was also detected by polymerase chain reaction. Nine tumours were located in the oropharynx (tonsillar area) and 27 in the larynx. RESULTS: HPV DNA was found in 14 of 25 (56%) laryngeal SCCs and in five of nine (56%) tonsillar SCC specimens examined; 17 out of the 19 HPV-DNA-positive cases showed high-grade IFI16 expression. Overall, proliferative activity was significantly related to tumour differentiation and histological grading. IFI16 protein expression was significantly inversely correlated with Ki67 (P = 0.039). Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index. CONCLUSIONS: To our knowledge, this is the first expression analysis of the IFN-inducible IFI16 gene in HNSCC. Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index.
High-risk human papillomavirus types, especially type 16, are risk factors for cervical cancer. Preliminary studies suggest that HPV16 polymorphisms in the long control region or in the E6 gene may alter the oncogenic potential of the virus. This could partially explain why some lesions progress to cancer while others do not. A systematic study combining the long control region and E6 has not been undertaken. This prompted us to investigate the long control region and the E6 in northern European women infected with human papillomavirus 16. We identified the sequence variations of both regions and investigated the long control region promoter activity among various isolates. In addition, we correlated the distribution of long control region and E6 polymorphisms with disease status. We analyzed 45 samples from Swedish and Finnish women. The long control region and the E6 gene were sequenced after polymerase chain reaction long control region fragments of six European isolates covering the majority of polymorphisms in this region were ligated into the pALuc vector and used for luciferase assays. In European HPV16 isolates, polymorphisms in the long control region are more frequent than in the E6 gene. Nevertheless, the promoter function was slightly increased in only one of the tested European long control region variants. In addition, we found a specific European E6 variant, L83V, to be enriched in high-grade lesions and cancer rather than a specific European long control region variant. The difference in oncogenicity between European HPV16 genotypes is more probably due to an altered property of the corresponding E6 proteins rather than to an altered activity of the P97 promoter.
Infection with high-risk human papillomavirus (HPV) is necessary for the development of a cervical lesion, but only a fraction of precursor lesions progress to cancer. Additional factors, other than HPV type per se, are likely to increase the probability for progression. Intratype genome variations have been reported to be associated with viral persistence and the development of a major cervical disease. We have recently shown that the prevalence of specific HPV16-E6 variants in invasive cervical cancer (ICC) varies between Italian and Swedish women. To extend our initial study we have analyzed E6 variants in cervical lesions from Czech women, ranging from low-grade cervical intraepithelial neoplasia (LCIN) to ICC and scaled up the sample size of our initial study of Swedish and Italian women. In addition, we have correlated the cases of cancers with human leukocyte antigen (HLA) class II haplotypes. In line with our earlier observation, the distribution of specific HPV16-E6 genotypes in CIN and ICC varied in the 3 cohorts. For instance, the HPV16-E6 L83V variant, which has been found to be positively associated with ICC in Swedish women (p = 0.002), was more prevalent in LCIN than in ICC in Italian and Czech women (p = 0.01 and = 0.03, respectively). These data indicate that host genetic factors, such as HLA polymorphism, may determine the potential oncogenicity of the HPV16-E6 L83V variant. Indeed, the DR04-DQ03 haplotype, which is approximately 3-fold more abundant in the normal Swedish population than in those in Italy and the Czech Republic, was found to be positively associated with HPV16-E6 L83V in the 3 cohorts investigated (p = 0.01). This observation may explain why L83V is a risk factor more in Sweden than in the other 2 countries.
Vaccination with oncogene-derived DNA for anti-cancer treatment carries a risk of de-novo tumor induction triggered by the persisting recombinant DNA. We hypothesized that an oncoprotein whose primary sequence has been rearranged ('shuffled') to maintain all possible T cell epitopes still induces cytotoxic T cells against the authentic protein but is devoid of transforming properties. As a model antigen, we used the E7 oncoprotein of the human papillomavirus (HPV) type 16, the major cause of cervical cancer. We have generated an artificial E7 molecule in which four domains were rearranged and, in order to maintain all possible T cell epitopes, certain sequences were duplicated. Upon transfection of this shuffled E7 gene (E7SH) into RMA cells, presentation of an E7 Db-restricted T cell epitope was shown by an E7-specific CTL line in vitro. Immunization of C57BL/6 mice with E7SH DNA induced E7-specific CTL and also conveyed protection against E7-positive syngeneic tumor cells. No transforming activity of E7SH DNA in NIH3T3 cells was detected, as determined by focus formation, induction of S-phase under conditions of serum deprivation and degradation of endogenous pRB. Our results suggest that DNA shuffling may become a promising concept for DNA-based anti-cancer vaccines.
Risk factors other than human papillomavirus (HPV) infection per se for cervical cancer development have been investigated recently. It was suggested that HPV 16 E6 variants and the p53 codon 72 arginine polymorphism could be progression markers. Indeed, it has been demonstrated that specific E6 variants and p53 arginine were both enriched in cancer. However, especially with regard to the latter, divergent results have been reported. Our aim was thus to investigate whether p53 arginine is important for cervical carcinogenesis by scaling up samples of the two European cohorts, the initial results of which were reported previously. In addition, we have assessed the occurrence of p53 codon 72 arginine, in combination with specific HPV 16 E6 genotypes. We found p53 arginine to be increased in cancer of both cohorts, consistent with our previous concept. Although specific E6 genotypes increased gradually with the severity of the lesion, p53 arginine was enriched in cancer only. Moreover, the frequency of the arginine allele was similar in groups with different E6 genotypes. It is concluded that p53 arginine is a risk factor for cervical cancer but probably acts independently of E6 variants.
A plate-binding assay was developed to quantify the affinity of the E7 oncoprotein from different human papillomavirus (HPV) types for the tumour suppressor pRb. The method is highly reproducible, sensitive and easy to handle. It could be easily adapted for the quantitative study of other interacting proteins and for screenings of inhibitors of protein/protein interactions. The pRb-binding affinity of six different E7 proteins has been quantified. The K(D) values vary from approximately 4.5x10(-9) M for HPV16 E7 to more than 1x10(-7) M for HPV10 and HPV48 E7. Point mutation C24G in the high affinity pRb-binding domain of HPV16 E7 results in a 3-fold affinity reduction. The data indicate that the high affinity pRb-binding domain of E7, LXCXE, is essential for the association between the viral and cellular proteins. However, other E7 domain(s), which appear(s) not to be present in all E7s, contribute to stabilize the E7-pRb association.
It has previously been shown that the E7 protein from the cutaneous human papillomavirus type 1 (HPV1), which is associated with benign skin lesions, binds the product of the tumor suppressor gene retinoblastoma (pRb) with an efficiency similar to that of the E7 protein from the oncogenic HPV type 16. Despite this ability, HPV1 E7 does not display any activity in transforming primary cells. In addition, the two viral proteins differ in their mechanisms of targeting pRb. HPV16 E7 promotes pRb destabilization, while cells expressing HPV1 E7 do not show any decrease in pRb levels. In this study, we show that HPV1 E7, in contrast to HPV16 E7, has only a weak activity to neutralize the effect of cyclin-dependent kinase inhibitor p16INK4a. By generation of HPV1/16 E7 chimeric proteins, we have identified a central motif in the two E7 proteins, which determines their different abilities to overcome the p16INK4a-mediated cell cycle arrest. This motif is located downstream of the pRb-binding domain and comprises only three amino acids in HPV16 E7. Swapping this central motif in the two viral proteins causes an exchange of their activities involved in circumventing the inhibitory function of p16INK4a. Most importantly, our data show that the efficiency of the E7 proteins in neutralizing the inhibitory effect of p16INK4a correlates with their ability to promote pRb degradation.
The HPV16 E7 oncoprotein neutralizes several cell cycle checkpoints, favouring the entry of quiescent cells into S phase. This activity is mediated in part by association of E7 with the pocket proteins and consequent activation of E2F transcription factors. In addition, HPV16 E7 protein is able to promote apoptosis. In this study we demonstrate that the ability to induce apoptosis is a common property of E7s belonging to both benign and malignant HPV types. The E7-induced apoptosis is mediated by inactivation of pRb, whilst neutralization of the other two pRB-related proteins, p107 and 130, is not sufficient to trigger apoptosis. Moreover, we show that certain point mutations in the conserved region 1 (CR1) of HPV16 E7 abolish the induction of apoptosis without altering the ability to stimulate S phase. Thus, these two E7-mediated cellular events, apoptosis and S phase entry, can be separated in immortalized rodent fibroblasts. Our findings demonstrate that the E7-mediated pRb destabilization is not required for its ability to drive quiescent cells into S phase and to induce apoptosis. Finally, expression of E7 proteins in NIH3T3, which lack a functional p19ARF, does not lead to p53 accumulation, indicating that the E7 impacts upon additional cellular pathways to promote apoptosis.
Whereas the tumor acidic extracellular pH plays a crucial role in the invasive process, the mechanism(s) behind this acidification, especially in low nutrient conditions, are unclear. The regulation of the Na(+)/H(+) exchanger (NHE) and invasion by serum deprivation were studied in a series of breast epithelial cell lines representing progression from non-tumor to highly metastatic cells. Whereas serum deprivation reduced lactate production in all three cells lines, it inhibited NHE activity in the non-tumor cells and stimulated it in the tumor cells with a larger stimulation in the metastatic cells. The stimulation of NHE in the tumor cell lines was the result of an increased affinity of the internal H(+) regulatory site of the NHE without changes in sodium kinetics or expression. Serum deprivation conferred increased cell motility and invasive ability that were abrogated by specific inhibition of the NHE. Inhibition of phosphoinositide 3-kinase by overexpression of a dominant-negative mutant or wortmannin incubation inhibited NHE activity and invasion in serum replete conditions while potentiating the serum deprivation-dependent activation of the NHE and invasion. These results indicate that the up-regulation of the NHE by a phosphoinositide 3-kinase-dependent mechanism plays an essential role in increased tumor cell invasion induced by serum deprivation.
Studies on human papillomavirus type 16 have demonstrated that the product of the early gene, E7, plays a key role in the immortalization and malignant transformation of the host cell. Several of the biological activities of HPV16 E7 are mediated by inactivation of the members of the pocket protein family, pRb, p107 and p130. In this study, we have characterized the in vitro properties of five E7 proteins from benign and malignant HPV types (10, 32, 48, 54, 77). We show that these E7 proteins associate with pRb and p107 with different efficiencies. All E7s increased the proliferative rate of immortalized rodent fibroblasts cultured in 10% calf serum containing medium. This property is completely independent of their ability to associate with the pocket proteins. Furthermore, all E7s, except HPV10 E7, stimulate G1/S progression and activated the cyclin E and cyclin A promoter in the absence of growth factors. This activity also does not correlate with the E7-efficiency of binding the pocket proteins. Together these data provide evidence that different E7s alter the regulation of the cell cycle by diverse mechanism(s). Finally, this comparative analysis of the different E7 proteins demonstrates that the oncogenicity of a HPV type is not determined by the ability of E7 to associate with the pocket proteins.
In this study we investigate the mechanism of intracellular pH change and its role in malignant transformation using the E7 oncogene of human papillomavirus type 16 (HPV16). Infecting NIH3T3 cells with recombinant retroviruses expressing the HPV16 E7 or a transformation deficient mutant we show that alkalinization is transformation specific. In NIH3T3 cells in which transformation can be turned on and followed by induction of the HPV16 E7 oncogene expression, we demonstrate that cytoplasmic alkalinization is an early event and was driven by stimulation of Na+/H+ exchanger activity via an increase in the affinity of the intracellular NHE-1 proton regulatory site. Annulment of the E7-induced cytoplasmic alkalinization by specific inhibition of the NHE-1, acidification of culture medium, or clamping the pHi to nontransformed levels prevented the development of later transformed phenotypes such as increased growth rate, serum-independent growth, anchorage-independent growth, and glycolytic metabolism. These findings were verified in human keratinocytes (HPKIA), the natural host of HPV. Results from both NIH3T3 and HPKIA cells show that alkalinization acts on pathways that are independent of the E2F-mediated transcriptional activation of cell cycle regulator genes. Moreover, we show that the transformation-dependent increase in proliferation is independent of the concomitant stimulation of glycolysis. Finally, treatment of nude mice with the specific inhibitor of NHE-1, DMA, delayed the development of HPV16-keratinocyte tumors. Our data confirm that activation of the NHE-1 and resulting cellular alkalinization is a key mechanism in oncogenic transformation and is necessary for the development and maintenance of the transformed phenotype.
A novel protein family (p14.5, or YERO57c/YJGFc) highly conserved throughout evolution has recently been identified. The biological role of these proteins is not yet well characterized. Two members of the p14.5 family are present in the yeast Saccharomyces cerevisiae. In this study, we have characterized some of the biological functions of the two yeast proteins. Mmf1p is a mitochondrial matrix factor, and homologous Mmf1p factor (Hmf1p) copurifies with the soluble cytoplasmic fraction. Deltammf1 cells lose mitochondrial DNA (mtDNA) and have a decreased growth rate, while Deltahmf1 cells do not display any visible phenotype. Furthermore, we demonstrate by genetic analysis that Mmf1p does not play a direct role in replication and segregation of the mtDNA. rho(+) Deltammf1 haploid cells can be obtained when tetrads are directly dissected on medium containing a nonfermentable carbon source. Our data also indicate that Mmf1p and Hmf1p have similar biological functions in different subcellular compartments. Hmf1p, when fused with the Mmf1p leader peptide, is transported into mitochondria and is able to functionally replace Mmf1p. Moreover, we show that homologous mammalian proteins are functionally related to Mmf1p. Human p14.5 localizes in yeast mitochondria and rescues the Deltammf1-associated phenotypes. In addition, fractionation of rat liver mitochondria showed that rat p14.5, like Mmf1p, is a soluble protein of the matrix. Our study identifies a biological function for Mmf1p and furthermore indicates that this function is conserved between members of the p14.5 family.
In eukaryotic cells arrested in S-phase, checkpoint controls normally restrain mitosis until after replication. We have identified an array of previously unsuspected factors that modulate this restraint, using transformed hamster cells in which cycle controls are known to be altered in S-phase arrest. Arrested cells accumulate cyclin B, the regulatory partner of the mitotic p34(cdc2) kinase, which is normally not abundant until late G(2) phase; treatment of arrested cells with caffeine produces rapid S-phase condensation. We show here that such S-phase checkpoint slippage, as visualised through caffeine-dependent S-phase condensation, correlates with rodent origin and transformed status, is opposed by reverse transformation, and is favoured by c-src and opposed by wnt1 overexpression. Slippage is also dependent on a prolonged replicative arrest, and is favoured by arrest with hydroxyurea, which inhibits ribonucleotide reductase. This last is a key enzyme in deoxyribonucleotide synthesis, recently identified as a determinant of malignancy. Addition of deoxyribonucleosides shows that rapid S-phase condensation is suppressed by a novel checkpoint mechanism: purine (but not pyrimidine) deoxyribonucleosides, like reverse transformation, suppress cyclin B/p34(cdc2) activation by caffeine, but not cyclin B accumulation. Thus, ribonucleotide reductase has an unexpectedly complex role in mammalian cell cycle regulation: not only is it regulated in response to cycle progression, but its products can also reciprocally influence cell cycle control kinase activation.
Swedish and Italian women with HPV 16-positive cervical disease were checked for codon 72 polymorphisms of p53. In both groups, arginine homozygotes were enriched in cancer compared with controls and precursor lesions.
Cell proliferation and terminal differentiation are mutually exclusive in most cell lineages. The b-zip transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) induces proliferation arrest and differentiation in many cell types, suggesting that both activities are linked. Here we show that C/EBPalpha-mediated proliferation arrest and differentiation pathways can be separated by the E7 oncoprotein of the "high-risk" human papilloma virus 16. The E7 oncoprotein overrides C/EBPalpha-mediated cell cycle withdrawal without compromising the transactivation activity of C/EBPalpha or its ability to participate in differentiation. Uncoupling of both pathways depends on the casein kinase II site of the oncoprotein but not on its ability to neutralize pocket proteins or the cyclin-dependent kinase inhibitor protein p21. Our results suggest a bifurcation of C/EBPalpha-mediated proliferation arrest and differentiation pathways.
High risk types of human papillomavirus (HPV) are agents in the aetiology of cervical carcinoma. The products of two early genes, E6 and E7, appear to be the principal transforming proteins. Studies of various monolayer cell culture systems have shown that the E7 oncoprotein of human papillomavirus type 16 is able to neutralize or bypass the inhibitory effect of the cell cycle-dependent kinase (CDK) inhibitors (CKIs) p21WAF1/CIP1 and p27KIP1. To understand whether the p21WAF1/CIP1 or p27KIP1 neutralization also plays a role in vivo, we performed studies on clinical specimens. Forty-five cervical biopsies, including HPV-negative mucosa, HPV 16-positive preinvasive (low and high grade lesions) and invasive neoplasia as well as HPV 6-positive condyloma acuminatum were analysed by single and double immunohistology. We examined the positive cell cycle regulator cyclin A and the universal cell cycle marker Ki67 as well as the negative cell cycle regulators p21WAF1/CIP1 and p27KIP1. Here, we show that in a significant fraction of cells the G1 block can be overcome despite high levels of CKIs in HPV lesions. This phenomenon, which was more evident for p21WAF1/CIP1 than for p27KIP1 was most marked in low grade lesions and in condylomata acuminata, in which a high viral productivity is expected. These results indicate that the overriding of CKI inactivation by viral oncoproteins appears to be a conserved property between low and high risk HPV types. We conclude that the CKI neutralization by HPVs is likely to be required for viral DNA replication rather than for malignant transformation of the host cell.
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