Miriam C. Guido

Asian-american variants of human papillomavirus type 16 have extensive mutations in the E2 gene and are highly amplified in cervical carcinomas

Human‐papillomavirus (HPV)‐E2 protein is involved in gene‐expression regulation and replication of HPV genome. Disruption of the E2 gene during viral integration has been proposed as a mechanism of tumoral progression, since the expression of E6/E7 viral oncogenes is allowed. However, retention of E1/E2 genes and high viral amplification are frequently found in HPV16‐positive carcinomas of some populations. In this study, we investigated whether retention of E1/E2 and viral amplification are associated with particular HPV16 E2 variants in cervical carcinomas. HPV16 detection, E1/E2 integrity and viral amplification were explored by Southern blot in 123 cervical carcinomas. HPV16 variants were identified by Southern blot and by sequencing E6, L1/MY and E2 regions. Of 46 HPV16‐positive tumors, 34 were positive for E1/E2 and 14 of them showed a variant restriction pattern by mutations in E2. All 14 were Asian‐American (AA) variants and, of 11 sub‐classified, 6 were AA‐a and 5 AA‐c. Two E1/E2‐negative tumors also contained the AA‐c variant, while the remaining HPV16‐positive tumors contained only European variants. The E2 gene of AA variants showed 24 mutations, 19 identical in both sub‐classes. The 24 mutations were distributed throughout the entire gene and 19 result in 18 amino‐acid changes. The AA variants were associated with E1/E2‐positive carcinomas with more than 50 viral copies/cell (p = 0.035). The association of Asian‐American E2 variants with retention of E1/E2 suggests that E2 variation may be an alternative mechanism de‐regulating the expression of viral oncogenes. Int. J. Cancer 83:449–455, 1999.

Early promoters of genital and cutaneous human papillomaviruses are differentially regulated by the bovine papillomavirus type 1 E2 gene product

The physical state of the human papillomavirus (HPV) genome is usually different in malignant lesions of the skin, in which it is generally found in episomal form, and genital mucosa, in which it is frequently integrated with disruption of the E2 gene. Using chimeric or natural HPV promoters in the presence of the bovine papillomavirus type 1 E2 gene product, we observed transcription activation or repression, depending on the distance of E2-binding motifs from the start site. We found a clear difference in the positions of E2-binding motifs in cutaneous and genital HPVs that may partly explain the selective pressure for genome integration of genital HPV types in malignant lesions.

Blood group O alleles in Native Americans: implications in the peopling of the Americas.

All major ABO blood alleles are found in most populations worldwide, whereas the majority of Native Americans are nearly exclusively in the O group. O allele molecular characterization could aid in elucidating the possible causes of group O predominance in Native American populations. In this work, we studied exon 6 and 7 sequence diversity in 180 O blood group individuals from four different Mesoamerican populations. Additionally, a comparative analysis of genetic diversity and population structure including South American populations was performed. Results revealed no significant differences among Mesoamerican and South American groups, but showed significant differences within population groups attributable to previously detected differences in genetic drift and founder effects throughout the American continent. Interestingly, in all American populations, the same set of haplotypes O(1), O(1v), and O(1v(G542A)) was present, suggesting the following: (1) that they constitute the main genetic pool of the founding population of the Americas and (2) that they derive from the same ancestral source, partially supporting the single founding population hypothesis. In addition, the consistent and restricted presence of the G542A mutation in Native Americans compared to worldwide populations allows it to be employed as an Ancestry informative marker (AIM). Present knowledge of the peopling of the Americas allows the prediction of the way in which the G542A mutation could have emerged in Beringia, probably during the differentiation process of Asian lineages that gave rise to the founding population of the continent.

Ha-ras oncogene–induced transcription of human papillomavirus type 18 E6 and E7 oncogenes

Human papillomavirus (HPV) DNA sequences are found in most carcinomas originating from the uterine cervix. HPV E6 and E7 oncogenes have been shown to cooperate with ras oncogenes to fully transform human epithelial cells. We investigated the effect of the Ha‐ras oncogene on the transcriptional activity of HPV‐18 and found that it induced the transcriptional activity of the viral promoter, whereas the normal gene had only a minimal effect. However, transfection of the normal Ha‐ras gene and simultaneous inhibition of protein phosphatase sensitive to okadaic acid (OA) resulted in a cooperative transactivation of the viral promoter. When cloned upstream of a minimal promoter, the AP‐1 binding sites present in the viral promoter conferred transcriptional responsiveness to Ha‐ras and OA. Furthermore, HeLa cell clones permanently expressing the Ha‐ras oncogene or high levels of the normal gene exhibited a twofold to threefold increase in E6*E7/E1 and E6*E7 transcripts. We propose that both Ha‐ras and a protein phosphatase sensitive to OA regulate HPV oncogene expression through modulation of AP‐1 activity and suggest that increased levels of E6 and E7, resulting from activated viral transcription in the presence of ras oncogenes, may in part explain the observed cooperation between these viral and cellular oncogenes in the transformation of human cells. Mol. Carcinog. 19:83–90, 1997.

Protocols for the Manual and Automatic Microinjection of Somatic Cells in Culture and for the Analysis of Microinjected Cells

Micromanipulation of living cells was first used in electrophysiology studies during the first half of this century. Reliable techniques for intracellular recording were developed in the late 1940s. Less than one decade later, similar techniques were applied to perform direct transfer of biological material into cells (see Chambers and Chambers 1961). Initially, microinjection was designed for transplantation of mammalian nuclei (Graessmann 1970) and chromosomes (Diacumakos 1973) into recipient cells in culture. Further refinement of materials and instruments, however, permitted intracellular transfer of solutions containing macromolecules of various origins, such as viral RNA (Graessmann and Graessmann 1976; Stacey et al. 1977), purified proteins (Mabuchi and Okuno 1977; Tjian et al. 1978), and viral DNA (Anderson et al. 1980; Capecchi 1980). Likewise, microinjection was used for DNA transfer into fertilized eggs and a cloned gene was soon reported to be expressed in mouse somatic tissues (Gordon et al. 1980). Later, recombinant genes were stably introduced into the mouse germ line (Brinster et al. 1981; Wagner et al. 1981). Since then, microinjection has been used routinely to generate transgenic animals, and applied to a wide variety of studies using living cells, such as gene expression, signal transduction, or cytoskeleton studies.

Immunization with Human Papillomavirus 16 L1+E2 Chimeric Capsomers Elicits Cellular Immune Response and Antitumor Activity in a Mouse Model.

Development of cervical cancer is associated with persistent infections by high-risk human papillomavirus (HPV). Although current HPV L1-based prophylactic vaccines prevent infection, they do not help to eliminate prevalent infections or lesions. Our aims were (i) to generate a vaccine combining prophylactic and therapeutic properties by producing chimeric capsomers after fusion of the L1 protein to different fragments of E2 from HPV 16, and (ii) to evaluate their capacity to generate an antitumoral cellular response, while conserving L1 neutralizing epitopes. Chimeric proteins were produced in Escherichia coli and purified by glutathione S-transferase (GST)-affinity chromatography. Their structure was characterized using size exclusion chromatography, sucrose gradient centrifugation, electron microscopy, and anti-L1 enzyme-linked immunosorbent assay. All chimeric proteins form capsomers and heterogeneous aggregates. One, containing part of the carboxy-terminal domain of E2 and its hinge region (L1Δ+E2H/NC, aa 206-307), conserved the neutralizing epitope H16.V5. We then evaluated the capacity of this chimeric protein to induce a cytotoxic T-cell response against HPV 16 E2. In (51)Cr release cytotoxicity assays, splenocytes from C57BL/6 immunized mice recognized and lysed TC-1/E2 cells, which express and present endogenously processed E2 peptides. Moreover, this E2-specific cytotoxic response inhibited the growth of tumors of TC-1/E2 cells in mice. Finally, we identified an epitope (aa 292-301) of E2 involved in this cytotoxic response. We conclude that the L1Δ+E2H/NC chimeric protein produced in bacteria can be an effective and economically interesting candidate for a combined prophylactic and therapeutic vaccine that could help eliminating HPV16-positive low-grade cervical lesions and persistent viral infections, thus preventing the development of lesions and, at the same time, the establishment of new infections.