Tesi di dottorato di ricerca
DI IESU, MARILENA
Role of MMTV-like virus in human breast cancer: determination of viral presence in a primary cell line.
Settore scientifico disciplinare
Corso di studi
SCIENZE CLINICHE E TRASLAZIONALI
tutor Prof.ssa Freer, Giulia
- breast cancer
Data inizio appello
Data di rilascio
Murine mammary tumor virus (MMTV) is an oncogenic beta-retrovirus discovered by Bittner in 1936, who demonstrated the involvement of the virus in the development of murine mammary cancer. It was shown to cause mammary carcinoma in mice following milk transmission from mother to suckling offspring. Oncogenesis occurs because the provirus integrates into the murine genome in proximity of regulatory gene promoters, modifying their expression. <br>Based on epidemiological observations, such a virus has also been hypothesized to be involved in the ethiology of certain types of human breast cancer. Indeed, MMTV viral sequences have been identified in DNA isolated from human mammary tumor cells, which also expressed the MMTV Env protein. Furthermore, sequences homologous to MMTV env have been observed in 38% of American patients with breast cancer. A sequence of 660 base pairs (bp) similar to MMTV env (env-like) was detected in tissue from such patients but not in normal breast tissue. These viral sequences showed a 95-98% homology to MMTV and only 56% homology with human endogenous retrovirus HERV-K10. This led to the hypothesis that there is a human virus similar to MMTV, the human mammary tumor virus (HMTV). <br>To assess HMTV involvement in the genesis of human breast cancer, this study was based on the analysis of a primary cell line taken from a patient with breast tumor, which was positive to HMTV genome by PCR. The aim of our research was to demonstrate the existence of such a virus, as whole virions, viral proteins or as a viral genome sequence, in this primary cell line. We attempted to confirm HMTV presence by searching for viral genome and its integration into the cell genome. In parallel, we tried to detect viral proteins such as Env and Pol, by different approaches.<br>Since the primary line in our hands appeared to progressively lose PCR positivity to HMTV genome, first of all, limiting dilution cloning was performed, in order to achieve positive and negative clones of the line itself. Positive and negative clones and the primary cell line were subjected to further analysis.<br>To assess whether viral proteins, such as Env were expressed in the cell line, Indirect Immunfluorescence analysis (IFA) was performed on clones and line with monoclonal antibodies against Env peptides. Viral expression was also determined in both clones and primary line by means of Western blot experiments, where the signal peptide of the Env protein, a protein of 14 kDa, was searched in cell lysates by means of monoclonal antibodies. While the presence of viral Env protein was found by IFA technique, Western Blot on p14 failed to detect the presence of Env. <br>In order to search for another viral protein in cell-free supernatants of cell line and clones, a semi-quantitative real-time PCR technique, Syber green-based PCR-enhanced reverse transcriptase (SG-PERT) assay was used to quantify reverse transcriptase (RT). During a one-step reaction, RT derived from retroviral particles will convert the MS2 RNA into cDNA and cDNA is subsequently quantified by qPCR amplification of the MS2 cDNA. SG-PERT has given ambiguous results, as the amount of RT activity detected in cell line and clone supernatants is at limit of resolution of the method. Low amount of RT expression would not be surprising since many oncoviruses are known not to produce large amounts of virions in the tissues where they induce cancer. <br>To demonstrate the presence of viral DNA in the genome of cell line and clones, PCR analysis was carried out. Indeed, a 220 bp fragment of HMTV env gene by PCR could be amplified from positive clones and line. To ascertain that viral DNA was integrated in cell genome and to identify the insertion point, Southern Blot, using as a probe a 600 bp sequence of MMTV env gene, was performed. It was not possible to identify the viral genome by Southern blot, but PCR amplification confirmed its presence. Again, such contradictory results are probably due to sensitivity differences in these two techniques. <br>Given the unclear results obtained, the next step was to search for the Gag protein. We therefore cloned two MMTV proteins that could be used to producing monoclonal antibodies to allow detection of viral Env and Gag proteins in the breast tumor line. Expression cloning of gp52 and p27 proteins of MMTV was carried out. For this purpose, the sequences coding for the two proteins were obtained from DNA of a mouse infected from MMTV and cloned in an expression vector that allowed the addition of a tail of 6 histidine residues. The 6-His tag could thus be detected by anti-histidine antibodies in Western blot and subsequently used to purify the recombinant proteins by affinity chromatography. These proteins were used to inoculate mice so as to obtain the aforesaid antibodies.<br>In an effort to determine the local prevalence of antibodies against MMTV, the recombinant proteins were also used to test patient sera for anti-HMTV antibodies in Western Blot. We analized 100 sera of normal individuals and 9 sera of women affected by breast cancer. Only 1% of the normal sera were positive to Gag p27 and Env gp52, whereas 33,3% (3/9) of sera from women affected by breast cancer positive to gp52 Env and 10% (1/9) positive to Gag p27 protein. These preliminary data suggest that this approach may bring very interesting results, therefore sera of patient with breast cancer will be collected and tested to obtain a statistically relevant number of cases. This approach may be used to test population for the presence of antibodies in sera for diagnostic purposes in the future.
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