apyrase and Papillomavirus-Infections

Cervical cancer is the second most frequent cancer in women in Mexico, and its development depends on the presence of human papillomaviruses in the uterine cervix. These oncogenic viruses transform cells where the control over cell cycle disappears, and the capacity to induce apoptosis is absent. On the other hand, some mutations confer to the transformed cells the ability to evade recognition by the immune system. The expression of markers of the immune system such as CD95, MICA/B, CD39, CD73, NKp30, NKp46, CD44, CD24, NKG2A, and CTLA-4 was analysed by flow cytometry on cervical cancer cells INBL (HPV 18, stage IVB), HeLa (HPV 18), CaSki (HPV 16), and C33A (HPV-). Our results showed the presence of atypical markers on cervical cancer cells; some of them are molecules involved in tumour cell recognition such as MICA/B and CD95. Other markers associated with immune system escape, such as CD39, CD73, and CTLA-4, were also present. Furthermore, we found that some cervical cancer cells expressed typical markers of NK cells like NKp30, NKp46, NKG2A, and KIR3DL1. It is not clear whether these molecules confer any gain to the tumour cells or if they represent a disadvantage, but we hypothesise that these molecules that are present in cervical cancer cells allow them to mimic in front of the immune system.

Topics: 5'-Nucleotidase; Antigens, CD; Apyrase; CTLA-4 Antigen; fas Receptor; Female; HeLa Cells; Histocompatibility Antigens Class I; Human papillomavirus 16; Human papillomavirus 18; Humans; Immunologic Surveillance; Killer Cells, Natural; Papillomavirus Infections; Receptors, Natural Killer Cell; Tumor Escape; Uterine Cervical Neoplasms

The development of cervical cancer (CeCa) is associated with high-risk human papilloma virus (HR-HPV) infections, mainly HPV-16, which is present in more than 50% of cases. The presence of immunosuppressive factors in the early stages of the disease is also strongly linked to CeCa progression. In this context, it is unknown whether ectonucleotidases CD39 and CD73, which are involved in the production of adenosine (Ado) that suppresses the specific antitumor immune response, are present in precursor lesions of CeCa. In this pilot study, we analyzed the presence of CD39 and CD73 and their capacity to generate Ado in 25 cervical samples from patients with grade 1 cervical intraepithelial neoplasms (CIN-1) and 25 samples from normal donors (NDs) free of HPV infection. Cells obtained from cervical samples of CIN-1 patients positive for HPV-16 showed higher CD39 and CD73 contents compared to samples obtained from CIN-1 patients negative for HPV-16 and NDs. Interestingly, solubilized cervical mucus from these patients also showed higher contents of soluble CD39 and CD73, which were associated with a greater capacity to produce Ado from the hydrolysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). In addition, serum samples of these patients showed higher levels of TGF-

Topics: 5'-Nucleotidase; Adenosine Monophosphate; Adenosine Triphosphate; Adolescent; Adult; Antigens, CD; Apyrase; Cross-Sectional Studies; Female; Human papillomavirus 16; Humans; Papillomavirus Infections; Transforming Growth Factor beta; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms; Young Adult

A method is described for HPV genotyping based on multiplex competitive hybridization (MUCH) combined with apyrase mediated allele-specific extension (AMASE). Two type-specific oligonucleotides were designed for each of the 23 investigated HPV types and directed towards two highly inter-type heterogeneous regions. The type-specific oligonucleotides were allowed to compete in the hybridization to an immobilized template resulting in a highly specific hybridization process. To increase further the specificity, a second step of type discrimination was used in which specific extension of 3'-termini matched oligonucleotides was performed. The 46 type-specific oligonucleotides each had a unique tag sequence to allow detection via an array of oligonucleotides complementary to the tags. To evaluate the genotyping assay, a total of 92 HPV positive samples were tested in this study. Twelve had double infections and five had three to five coexisting HPV types. The results show that MUCH-AMASE can readily detect multiple infections, whereas conventional dideoxy sequencing resulted in ambiguous sequence. Four samples with three to five genotypes detected were cloned and individual clones were sequenced. The cloning procedure verified the MUCH-AMASE results with indications that we can find minor infections (<2% relative amounts). We can thus conclude that the developed assay is highly sensitive, with improved throughput and with excellent possibility to detect multiple infections.

Topics: Alleles; Apyrase; DNA Primers; Female; Gene Expression Profiling; Humans; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Papillomaviridae; Papillomavirus Infections; Sensitivity and Specificity; Species Specificity; Templates, Genetic