The effects of HIV-1 Tat protein on cell cycle during cervical carcinogenesis.

The role of HPV in the carcinogenesis of intraepithelial and invasive anogenital lesions is currently well established. E6 and E7 oncoproteins of high-risk HPV genotypes are known to inactivate p53 and pRb pathways. Several studies have described an increased prevalence and recurrence of both cervical HPV infection and invasive cervical cancer among HIV-1 positive women compared to HIV-1 negative cases. For these reasons, cervical cancer is considered an AIDS-defining neoplasm. Unlike other AIDS-associated neoplasms, the occurrence of cervical cancer is independent of immune suppression. HIV-1 infection in patients with high grade precancerous lesions and invasive cervical cancers results in a therapy refractory and more aggressive disease phenotype, which is not yet well understood at the molecular level. An upregulation of HPV E6 and E7 gene expressions by HIV-1 proteins such as Tat has been documented by some authors. However, the role of HIV-1 in cervical carcinomas is still unclear. It is already known that HIV-1 Tat protein is able to influence cell cycle progression. Altogether, these facts led us to investigate the effects of Tat on the expression of cell cycle regulator genes. After transfection of HeLa cells with Tat, we analyzed the expression of cell cycle regulators from these cells by IHC and Real-time PCR. A significant reduction in the expression of cell cycle inhibitors of transcription and an increase in the levels of proliferation markers were observed. These results suggest that HIV-1 may enhance cervical carcinogenesis by promoting cell cycle progression. We also found that this HIV-1 Tat-induced cell proliferation was not dependent on the E2F family of transcription factors, and therefore postulate that Sp factors may be involved.

Different effects of interferon-alpha on melanoma cell lines: a study on telomerase reverse transcriptase, telomerase activity and apoptosis.

BACKGROUND: Although the antiproliferative and proapoptotic effects of interferon (IFN)-alpha are widely recognized, its antitumour mechanisms are not completely known. Recent studies indicate that the derepressed expression of the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), and telomerase activity (TA) are involved in the process of human carcinogenesis. Only a few studies have investigated the effects of IFN-alpha on hTERT and TA, with controversial results. Objectives To study the hTERT mRNA expression, TA and apoptosis in human melanoma cells treated with IFN-alpha. METHODS: Five human melanoma cell lines (Me665/2/21, Me665/2/60, HT-144, SK-Mel-28 and SK-Mel-5) were cultured in standard conditions and treated with 20000 IU mL-1 of human recombinant IFN-alpha-2b. Apoptosis was evaluated as hypodiploid DNA content determined by flow cytometry, caspase-3/7 activity by enzymatic assay, and poly(adenosine diphosphate-ribose) polymerase cleavage by Western blot analysis. IFN-alpha receptor (IFNA-R) and hTERT mRNA expression levels were evaluated by semiquantitative reverse transcription-polymerase chain reaction. TA was evaluated by a polymerase chain reaction-based telomerase repeat amplification protocol assay. RESULTS: Besides a variable degree of cell proliferation inhibition in all cell lines tested, we found different responses, ranging from no significant effects in SK-Mel-28 cells, to a high degree of apoptosis with no hTERT mRNA expression and TA modification in HT-144 cells, and induction of apoptosis, along with decrease in hTERT mRNA expression and TA in Me665/2/21 cells. No induction of apoptosis was observed in SK-Mel-5 and Me665/2/60 cells, although an early decrease in hTERT mRNA expression, and a minor increase of both hTERT mRNA expression and TA were found, respectively. CONCLUSIONS: Our results suggest that the effects of IFN-alpha on hTERT and TA can result from the induction of apoptosis, but they can also occur through a direct modulation of hTERT. We hypothesize that, depending on the cellular context rather than the IFNA-R status of the targeted cells, IFN-alpha can elicit an apoptotic cell death; furthermore, different pathways of apoptosis, not necessarily involving telomerase, can be put into motion.

Telomerase activity, Ki-67, cyclin D1 and A expression, and apoptosis in solitary fibrous tumors: additional features of a predictable course?

Solitary fibrous tumors (SFTs) are infrequent soft tissue neoplasms which are usually benign and surgically curable. However, their behavior is not always predictable, although several clinical and pathological criteria of malignancy have been established. In many cancers, including some soft tissue tumors, telomerase activity (TA) has been shown to be a new reliable pathological marker of malignancy. Overexpression of some cyclins is associated with higher degrees of malignancy and predictive of the clinical course. In this study, we evaluated TA, mitotic and apoptotic indices (MI, AI), and the expression of Ki-67, cyclins D1 and A in five typical and two clinicopathologically atypical SFTs, the latter two of which had also recurred. High TA was demonstrated in the two atypical cases, which also showed a higher labeling index to Ki-67, as well as higher cyclin D1 and A expression, and either none or very few apoptoses. We suggest that TA, Ki-67, cyclin expression, and AI be evaluated in SFTs as possible adjunctive pathological criteria of behavior.

Evaluation of telomerase activity in cutaneous melanocytic proliferations.

Telomerase is an enzyme which synthesizes the telomeres, TTAGGG repeats at the end of vertebrate chromosomes. Its activity is suppressed in the majority of somatic cells, whereas it is detectable in most tumor cell lines and human cancers. Telomerase activity has been evaluated in many tumors for diagnostic purposes, and an increase thereof has been found with tumor progression. In our study we used anonisotopic polymerase chain reaction (PCR)-based TRAP (telomeric repeat amplification protocol) method to quantify the level of telomerase activity in a series of cutaneous melanocytic lesions. Thirty-three benign nevi, 8 dysplastic nevi, 38 malignant melanomas, and 4 melanoma metastases were analyzed. Mean relative telomerase activity was low in benign nevi (3.5+/-2.9), and significantly increased in dysplastic nevi (13.1+/-6.8), malignant melanomas (49.8+/-29.6), and metastases (121.2+/-11.2). In addition to the evaluation of telomerase activity as a possible diagnostic tool, its increase with tumor progression also suggest a prognostic role in cutaneous melanoma.

Detection of telomerase activity and correlation with mitotic and apoptotic indices, Ki-67 and expression of cyclins D1 and A in cutaneous melanoma.

Telomerase plays a key role in carcinogenesis. It is activated in most immortal cell lines and human cancers, including cutaneous melanoma (CM). Increased cell proliferation and deregulation of the cell cycle occur in human cancers. Links between telomerase activity (TA), cell proliferation, cell death and expression of cell-cycle regulators have not been extensively elucidated in CM. In this study, we investigated TA, mitotic index (MI), apoptotic index (AI), Ki-67 and nuclear positivity of cyclins D1 and A (Ki-67+ N/1,000, cyclin D1+N/1,000, cyclin A+N/1,000) in 42 primary cutaneous melanomas (PCMs). TA was detected in all cases and directly correlated with MI, Ki-67+N/1,000, cyclin D1+N/1,000 and cyclin A+N/1,000 (p < 0.001); it was not correlated with AI. When subdividing PCMs into radial and vertical growth phase melanomas (RGPMs, VGPMs), a correlation was maintained only with MI (p < 0.005) and cyclin D1 +N/1,000 (p < 0.005). Although MI and Ki-67+N/1,000 were highly correlated with cyclin D1+N/1,000 and cyclin A+N/1,000 (p < 0.001) when considering all cases together, a high correlation was found in the RGPM and VGPM groups between cyclin A+N/1,000 and Ki-67+N/1,000 only (p < 0.001), thus suggesting that cyclin A is more closely correlated with cell proliferation than cyclin D1. Our results further support the association between TA, tumor cell proliferation and cyclin D1 and A expression in PCM, though it is possible that links between TA and proliferation, on the one hand, and TA and cyclin D1 expression, on the other, might occur following various pathways.

Most Helicobacter pylori-infected patients have specific antibodies, and some also have H. pylori antigens and genomic material in bile: is it a risk factor for gallstone formation?

Bile may contain a 130-kDa protein endowed with aminopeptidase activity and the ability to promote cholesterol crystallisation. As >90% of H. pylori strains have a similar peptidase activity, and half the isolates express a 110- to 140-kDa antigen, the CagA protein, we investigated a possible association between H. pylori infection and gallstones, and the presence in bile samples of factors related to H. pylori that could increase cholesterol crystallization. The prevalence of H. pylori infection was 82.1% in 112 patients with gallstones and 80.3% in 112 controls (NS). Fifteen bile samples out of 23 specimens from infected patients (65.2%) contained anti-CagA antibodies. A approximately 60-kDa antigen only reacting with an anti-CagA antibody was found in five bile samples (21.7%) from 23 infected patients. One bile sample (4.1%) contained ureA and cagA genes of H. pylori. The homology of CagA with the N-terminal sequence of aminopeptidase N was very low. We concluded that the presence of specific antibody to H. pylori in most bile samples tested and of an H. pylori putative antigen in a discrete number of cases may represent factors that increase the risk of gallstone formation.

Abortive mitoses and nuclear DNA fragmentation in CD30+ large cells of Hodgkin's disease.

This study was undertaken to better comprehend the reasons for the scarcity of Hodgkin and Reed-Sternberg (H-RS) cells in Hodgkin's disease (HD) despite their expression of "proliferation-associated antigens". To this end, we assessed the relative frequency of mitotic phases and nuclear damage (detected by in situ end-labeling of DNA strand breaks) in CD30+ large cells of nodular sclerosis and mixed cellularity HD. Our results show that a) most CD30+ cells in HD exhibit abortive mitoses, with a highly significant arrest at the metaphase-ana/telophase transition, and b) many of these elements, i.e. mainly H-RS cells, show fragmentation of nuclear DNA, suggesting imminent or actual death. Percentages of CD30+ cells that entered mitosis and those with DNA strand breaks were of a similar order of magnitude and correlated significantly in a linear fashion. These findings are consistent with the concept that cell deletion is the major cause of the paucity of H-RS cells in HD.

Growth vs. DNA strand breaks in Hodgkin's disease: impaired proliferative ability of Hodgkin and Reed-Sternberg cells.

We re-appraised the cell renewal pattern in Hodgkin's disease (HD), considering that most, though not all, Hodgkin/Reed-Sternberg (H-RS) cells exhibit abortive mitoses and that a substantial fraction of these exhibits DNA damage suggestive of imminent or actual cell death. Using combined immunohistochemistry and in situ end-labeling to detect strand breaks, the percentage per case of CD30+ (mainly H-RS) cells with DNA fragmentation (DNA fragmentation index [DFI]) was estimated. For each case, we registered the mitotic index (MI) of CD30+ cells and the percentage of Ki-67+ atypical large cells. To quantify the sum of our parameters for mitosis, whether successful or not, and DNA damage, we introduced the kinetic event index (KEI = MI + DFI). Only DFI and KEI distinguished significantly between mixed cellularity and nodular sclerosis HD. The values for MI and DFI, and therefore for KEI, CD30+ and CD30- small lymphoid cells were proportional. The percentages of Ki-67+ large atypical cells (median 50%) did not correlate significantly with either MIs or DFIs of CD30+ cells. Cluster analysis revealed the existence, independent of histological subtype, of 2 large groups of HD with different KEIs. Our findings suggest that cell deletion plays an important role in HD. Further, it appears that proliferation-associated antigens in H-RS cells do not reflect successful cell production in this disorder.

Epstein-Barr virus infection in sinonasal non-Hodgkin's lymphomas.

Sinonasal non-Hodgkin's lymphomas (SNHLs) of B- or T-cell immunophenotype have been associated with Epstein-Barr virus (EBV) infection of neoplastic lymphoid tissue. Nine SNHLs were investigated using immunohistochemistry, the polymerase chain reaction (PCR) for EBV genome and in situ hybridization (ISH) for EBV encoded RNAs (EBER), immunoglobulin (CI-gHR) and clonal T-cell receptor (CTC beta R) gene rearrangements. Eight cases were diagnosed as peripheral pleomorphic T-cell lymphomas (pPTCL). PCR showed the presence of EBV genome in eight cases; ISH for EBER led to the detection of positive cells in five cases. Late membrane protein (LMP) immunostaining was observed in three cases. No EBV positivity has been detected in control cases. The frequent association with EBV infection in the cases illustrated confirms the previous suggestions that EBV may have a role in the genesis of lymphomas of the sinonasal region.

Distinction between diffuse cutaneous malignant follicular center cell lymphoma and lymphoid hyperplasia by computerized nuclear image analysis.

The difficult differential diagnosis between the diffuse variants of cutaneous lymphoid hyperplasia (CLH; synonym; pseudolymphoma) and malignant follicular center cell lymphomas (FCCL) often requires a multidisciplinary approach. Eighteen CLH and 11 FCCL, diagnosed by conventional histology and immunophenotyping and subsequently examined with a polymerase chain reaction to show clonal immunoglobulin heavy-chain gene rearrangements, were subjected to a novel type of automated nuclear image analysis. Of all nuclear parameters tested in azure A-stained semithin sections, the mean nuclear profile area (TN) of lymphoid cells was the best criterion to distinguish between CLH and FCCL (p = 9 x 10(-6)). Additional distinctive features, in the order of decreasing significance, were the SD of TN; all chromatin textural parameters combined; and the light and the dark fractions of the central nuclear profile areas. Parameters related to the chromatin pattern were independent of nuclear profile size in FCCL, but not in CLH. Two lesions registered as CLH displayed the nuclear characteristics favoring this diagnosis, but showed B-cell monoclonality at the DNA level. In conclusion, computerized nuclear image analysis is a helpful additional diagnostic tool in the evaluation of diffuse CLH and cutaneous FCCL.

Apoptotic index: discriminant feature for the differentiation of cutaneous diffuse malignant follicular center cell lymphomas from lymphoid hyperplasia.

Diffuse subtypes of cutaneous lymphoid hyperplasia (CLH; n = 18) and primary malignant follicular center cell lymphoma of the skin (FCCL, n = 11) were diagnosed by conventional histology, immunophenotyping on paraffin sections, and gene rearrangement analysis. We then counted on semithin, Azur A-stained sections of resin-re-embedded biopsy specimens the relative numbers of apoptotic bodies among all lymphoid cells (apoptotic index [AI]). The diagnostic value of AI was compared to that of mitotic indices (MI) and percentages of various cell types in the cutaneous infiltrate. Features of cellular infiltrates distinguishing to two groups of lesions, in the order of decreasing significance, were percent large lymphoid cells, percent medium-sized lymphoid cells (both higher in FCCL); percent small lymphoid cells, percent epithelioid/giant cells, and percent histiocytes/macrophages (all three higher in CLH). However, of all parameters tested, AI had the greatest discriminant value (median in FCCL 1.11%, in CLH 0.14%; p = 8 x 10(-6)). Two cases, diagnosed as CLH with all morphologic and immunologic methods used, showed B-cell monoclonality at the DNA level. Linear discriminant analysis determined the following order of distinctive power of variables: 1) AI; 2) MI; 3) percent small lymphoid cells; 4) percent medium-sized lymphoid cells; 5) percent large lymphoid cells; 6) percent epithelioid/giant cells; and 7) percent histiocytes/macrophages. The present study thus establishes AI as an important parameter in the differentiation of diffuse CLH from diffuse cutaneous FCCL.

Epstein-Barr virus and gastric cancer: data and unanswered questions.

Sixty-five unselected cases of gastric cancer have been analysed for EBV DNA by polymerase chain reaction, in situ hybridization and immunohistochemistry for CD21 antigen expression. Four cases were found EBV-positive by PCR, while ISH yielded positive results in 3 of these cases, demonstrating EBV in the nuclei of cancerous cells. CD21 antigen was expressed in cancerous cells in all 3 ISH-positive cases. All the EBV-positive cancers of the present series were poorly to moderately differentiated adenocarcinomas with prominent lymphoid infiltration. These results are discussed also on the basis of the literature.

Factor VIII:c concentrate virus inactivated: progress in purification by using classic chromatographic methods.

This study was carried out with the aim of developing a production process for the manufacture of a highly purified factor VIII concentrate which is virus inactivated by pasteurization in liquid phase. Beside standard plasma protein separation techniques, the procedure uses a chromatographic step on anion exchanger, whose selectivity is increased by using high, but not destabilizing salt concentrations. The final product before stabilization has a specific activity higher than 300 IU/mg of protein, namely the highest specific activity reported for human factor VIII concentrates purified without the use of immunoadsorbents.

Identification of monoclonal B-cell populations in lymphoid infiltrates of lacrimal gland by rapid cycle polymerase chain-reaction.

Lymphoid hyperplasia of lacrimal gland may be difficult to be differentiated from lymphomas on the basis of morphology and immunohistochemistry. The results of this study indicate that polymerase chain reaction should be employed for confirming the diagnosis of lymphoma in cases with histological and immunophenotypical characteristics of lymphomas, and for detecting monoclonal lymphoid cells in an otherwise non-lymphomatous but dubious or borderline morphological context.

Correlation between differentiation and lung colonization by retinoic acid-treated F9 cells as revealed by the expression pattern of extracellular matrix and cell surface antigens.

For study of the correlation between differentiation and organ colonization properties of tumor cells, F9 embryonal carcinoma (EC) cells were treated with retinoic acid, an inducer of differentiation; and their organ colonization pattern was assessed by the experimental metastasis assay. Untreated cells were found to colonize the liver, whereas treated cells colonized the lungs. This pattern held true when metastases were scored after spontaneous death or after a careful microscopic search for micrometastases. Histologic examination revealed that both the tumor nodules produced by the untreated and the treated cells had the characteristics of EC devoid of any evidence of differentiation. The immunohistochemical study of the expression of markers typical of embryonal carcinoma cells or of the extracellular matrix components laminin and collagen type IV, typical of differentiated cells, confirmed these results. However, the lack of expression of stage-specific embryonal antigen 1 (SSEA-1), a marker generally associated with the undifferentiated state, observed only in the tumors obtained after injection of treated cells, indicates that the lung nodules probably derive from cells that have responded to the induction in vitro but have dedifferentiated in vivo.

Organ colonization pattern of retinoic acid-treated and -untreated mouse embryonal carcinoma F9 cells.

The mouse embryonal carcinoma cell line F9 differentiates into parietal endoderm cells after a 3-day exposure to retinoic acid and dibutyryl cyclic AMP. Using the experimental metastases assay, we investigated the organ colonization properties of RA-treated and -untreated populations of F9 cells. The results show that untreated F9 cells colonize the liver with a high degree of specificity while the treated populations colonize the lungs. Cells derived from a lung colony colonized only the liver unless they were treated with RA. However, removal of the inducer from culture of differentiated cells did not cause reversal of the lung colonization potential. Our observations also indicate that it is unlikely that lung colonization is due to cell aggregation or to interaction between differentiated and undifferentiated cells. Taken together, these results suggest that RA induces the observed changes of organ colonization properties of F9 cells.