Maria Luisa Marcante

Human papillomavirus in breast cancer

SummaryHistological sections from paraffin-embedded breast carcinoma and axillary lymph nodes were examined for the presence of human papillomaviruses by two different techniques: the polymerase chain reaction (PCR) and thein situ hybridization with biotin-labelled probes. By PCR we detected HPV 16 DNA sequences in 29.4% of breast tumours and in some metastatic lymph nodes, though we were unable to identify any HPV DNA sequences byin situ hybridization. These results suggest that HPVs could play a role in the genesis of breast neoplasia.

Physical state and expression of human papillomavirus in laryngeal carcinoma and surrounding normal mucosa.

Epidemiologic and biomolecular evidence suggests that human papillomavirus (HPV) infection may be associated with the development of head and neck cancers. To clarify the role of HPV in larynx carcinoma, 25 patients were studied for the presence of viral DNA, possible virus integration into the cellular genome, and viral expression both in neoplastic tissues and in neighbouring normal mucosa. Twelve of 25 patients with neoplasia (48%) showed negative results for HPV sequences, and 13 (52%) showed positive results. Among the latter group of patients, seven were HPV‐16 positive, five were HPV‐6, and one was HPV‐45. No multiple infections were detected. The physical status of the HPV genome was analysed by three methods: polymerase chain reaction (PCR), bidimensional agarose gel electrophoresis, and in situ hybridisation. Viral integration into the host genome occurred in 43% of cases of HPV‐16 and in 20% of cases of HPV‐6. Viral RNA expression was detected by reverse transcription–PCR only in HPV‐16‐positive tumours. The pattern of expression was consistent with an active role of HPV in cellular transformation. In conclusion, the present work suggests that HPV infection may be involved in some cases of laryngeal carcinoma. However, the transformation mechanisms might be different from those currently accepted for anogenital cancers. J. Med. Virol. 60:396–402, 2000.

Tyrosinase protects human melanocytes from ROS-generating compounds

The effects of two tetrahydroisoquinolines (TIQs), tetrahydropapaveroline (THP) and salsolinol (SAL), on human primary melanocytes were studied. These compounds are naturally occurring alkaloids deriving from the condensation of dopamine with aldehydes. The effects on the viability were studied by treating the cells with variable concentration of THP or SAL; both TIQs were well tolerated up to roughly 30 micro M. At higher concentrations, THP became overtly toxic while SAL showed no cytotoxic effect up to 100 micro M. TIQs treatment determined an impairment of intracellular activity of antioxidant enzymes, like SOD, DT-diaphorase, and glutathione peroxidase. A decrease of alpha-ketoglutarate dehydrogenase activity was also evidenced following TIQs treatment; a very strong diminution was found in THP-treated cells, whose viability was highly decreased. Both TIQs increased tyrosinase-specific mRNA transcription followed, in the case of SAL, by an activation of tyrosinase. In the presence of tyrosinase inhibitors TIQs treatment resulted in a sharp cytotoxic effect even at concentrations normally well tolerated. Taken together these data suggest that tyrosinase represents an outstanding protective mechanism against ROS-generating compounds, once primary detoxifying mechanisms are impaired or not available.

Cytotoxicity of dopamine-derived tetrahydroisoquinolines on melanoma cells.

Tetrahydroisoquinolines (TIQs) are endogenous alkaloid compounds detected in urine, central nervous system and some peripheral tissues in Mammalia. No data are at present available on TIQ levels in skin, although in vitro biochemical evidences indicate that they may undergo auto-oxidation with production of reactive oxygen species or may be enzymatically converted into melanin pigments. The effect of two catechol-bearing TIQs, salsolinol (SAL) and tetrahydropapaveroline (THP), on the viability of melanotic or amelanotic melanoma cell lines was investigated. Both SAL and THP were well tolerated up to roughly 30 microM and became overtly toxic at higher concentrations, with SAL being better tolerated than THP. Intracellular activity of some antioxidant enzymes, tyrosinase and alpha-ketoglutarate dehydrogenase was also evaluated to assess the cell response to oxidative and metabolic challenge of TIQs treatment. Catalase and superoxide dismutase pre-treatment only partially prevented TIQs toxicity while a complete protection was obtained with N-acetylcysteine and GSH. TIQs were able to provoke upregulation of the scavenging enzymes catalase and DT-diaphorase and to determine a decrease of the alpha-ketoglutarate dehydrogenase activity. SAL and THP enhanced tyrosinase activity and melanin production, suggesting that they were indeed tyrosinase substrates leading to melanin formation. The results support the evidence that TIQs were toxic toward melanoma cells, leading to their death by necrosis. TIQs toxicity was likely due to increased oxidative stress by generation of reactive oxygen species and oxidative metabolites. Our study represents an intent to furnish an additional contribution for the comprehension of catechol cytotoxicity.

HPV 16 E7 antibody levels in cervical cancer patients: Before and after treatment

Antibody response to HPV16 E7 oncoprotein may represent a marker of cervical cancer. A HPV16 GST‐E7 fusion protein was used in a Western Blot assay to analyse the HPV16 E7 antibody response in 30 patients before and after treatment for cervical carcinoma (stage IIB or IIIB). Patients were treated with three courses of cisplatin/bleomycin therapy followed by surgery, or with surgery alone. Thirteen out of 30 patients had serum antibodies to HPV16 E7 antigen. Three months after chemotherapy little or no change in antibody titre was detected. In contrast, after surgery, a significant decrease in antibody titre was observed in 9/10 patients. In two cases the titre declined to zero 3 and 9 months after treatment, respectively. These results confirm the usefulness of studying anti‐HPV16 E7 antibody profile in cervical cancer patients and suggest that the serum response correlates with tumour burden. J. Med. Virol. 54:192–195, 1998.

INTERFERON-BETA -STRONG CYTOPATHIC EFFECT ON HUMAN PAPILLOMAVIRUS TYPE 16-IMMORTALIZED HPK-IA CELL LINE, UNEXPECTEDLY NOT SHARED BY INTERFERON-ALPHA

We report a novel, unusually severe cytopathic effect of interferon-beta (IFN-beta). Data concerning antibody neutralization, induction and recovery time course, CPE50 dose, impact on oxidative metabolic activity and 1D SDS-PAGE total cellular protein analysis are provided for preliminary characterization. This cytopathic effect appears to be linked to human papillomavirus type 16 (HPV-16) genome presence as it is markedly evident in the HPV-16-immortalized HPK-IA cell line, but is not induced in diploid keratinocytes. It is also not induced in highly malignant SiHa cells suggesting that it also requires a fairly conserved phenotype. This effect is unexpectedly not shared by IFN-alpha pointing to a discrimination between IFN-alpha and -beta signal despite the well-known sharing of a common receptor. It remains to be clarified whether this divergence, undetectable in other cellular systems, represents a direct effect of viral presence or a non-specific consequence of cellular homoeostatic disregulation induced by the papillomavirus genome.

Detection and typing of human papillomavims by single hybridization

A rapid and non-radioactive molecular hybridization test was developed which simultaneously detects and types different human papillomaviruses (HPV) DNA in fresh and paraffin-embedded clinical specimens. The method includes reverse blot hybridization between different recombinant HPV plasmids immobilized on nylon membrane and probe of cellular DNA amplified and biotin- or digoxigenin-labeled by the polymerase chain reaction (PCR). PCR protocol using consensus primers includes the mixing of Taq polymerase at high temperature (Hot-Start) and the addition of the hapten-conjugated nucleotide after the first ten cycles of amplification. The sensitivity level of this method resulted in detecting about 50 copies of HPV 16 for sample, independently of hapten used. The specificity of the typing method was also validated by more laborious and conventional analyses such as Southern-blot or PCR followed by several hybridizations with specific probes. Using this test HPV-11, -16, -18, -31 and -35 were typed in a number of samples from patients attending hospital. The method appears suitable for the handling of clinical samples in a selected population screening for type specific infections by HPV.

Human papillomavirus DNA as a possible index of invasiveness in female genital tract carcinomas

Paraffin-embedded tumour sections were used for the polymerase chain reaction (PCR) with three primer sets that amplify specific regions of human papillomavirus (HPV) types 11, 16 and 18. The positive samples were confirmed by hybridisation of the amplified sequences with the specific HPV probes. In all screened metastases the same viral sequences were found as in the primary tumour. HPV 16 was the most frequently detected virus in genital tract tumours. In a metastatic ovary carcinoma with unknown primary site HPV 16 DNA was observed. Moreover, pelvic lymph nodes with no microscopic evidence of metastases contained HPV DNA of the same subtype as the primary tumour. Thus, the HPV DNA detected by PCR is a useful indicator of neoplastic cells in the earlier stages of invasiveness. The finding of specific HPVs in the metastatic lesions could also provide information about the location of the primary neoplasia.

Primary Keratinocytes Can Be Infected by Natural Isolates of Genital Human Papillomavirus

Viral particles of human papillomavirus (HPV) types 11 and 16 were isolated from flat condylomas and biopsies of dysplastic lesions and used to infect human keratinocytes. The presence of HPV DNA in infected cells was determined by molecular hybridization and the polymerase chain reaction. Late transcripts were detected shortly after infection. The persistence of HPV was limited to early passages after infection and the loss of viral DNA preceded cell culture senescence. Our experimental evidence supports the idea that pooling of several lesions can lead to the isolation of infectious HPV 16 virions.