Loss of p14<Superscript>ARF</Superscript> expression in melanoma

Lack of p14ARF expression or its functional inactivation has been observed in human and murine carcinomas. Although very few mutations of p14ARF have been detected in some cancer types, changes in expression seem to play an important role in the development of other human cancers such as mesotheliomas. To examine the p14ARF gene and expression of p14ARF protein in melanomas, we screened eight human melanoma cell lines and primary human melanocytes by RT-PCR, sequencing and immunoblotting. All melanoma cell lines analyzed expressed wild-type p14ARF mRNA as well as protein. P14ARF expression was investigated by immunohistochemical staining of 32 tissue samples of benign melanocytic nevi (n=14), melanomas (n=12) and melanoma metastases (n=6). In contrast to the results obtained from cell lines in vitro the immunohistochemical stainings revealed a correlation between the progression of melanoma and the lack of the p14ARF protein expression. Positive p14ARF protein staining was observed in 11 of 14 benign nevi, in 3 of 12 melanomas and in 0 of 6 melanoma metastases. In summary, we demonstrated a significant inverse correlation between p14ARF protein expression and progression of melanocytic tumors since the amount of p14ARF protein staining decreased from benign melanocytic nevi to metastatic melanoma in situ. These results suggest that p14ARF inactivation is important in the development of melanomas.     

Bcl-2 and bcl-xL antisense oligonucleotides induce apoptosis in melanoma cells of different clinical stages

Recent clinical studies have shown the promise of bcl-2 antisense therapy in patients with melanoma. To further demonstrate the importance of bcl-2 and validate the related antiapoptotic protein bcl-xL as targets for antisense therapy in melanoma, their implication as survival factors in melanoma cells of different clinical stages as well as in normal melanocytes was investigated. Primary cell cultures derived from 17 melanomas, the cell line A375, and normal melanocytes from healthy donors were treated with antisense oligonucleotides targeting either the bcl-xL mRNA or the bcl-2 and the bcl-xL mRNAs simultaneously. Bcl-2 and bcl-xL expression in cells was analyzed by real-time polymerase chain reaction and Western blotting. Cell viability was assessed in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and apoptosis assays. Bcl-2 expression was low in melanoma cells of stages I, II, and III, hardly detectable in A375 cells, but high in normal melanocytes. Bcl-xL expression was high in all cell types tested. As shown in A375 cells and the stage III melanoma cells 0513, both the bcl-xL monospecific oligonucleotide 4259 and the bcl-2/bcl-xL bispecific oligonucleotide 4625 effectively reduced tumor cell viability by induction of apoptosis with IC50 values ranging from 200 to 350 nM. Oligonucleotide 4625 proved to be superior to 4259, as it significantly reduced the viability of cells from all melanoma stages. Both oligonucleotides reduced also the viability of normal melanocytes. Our data suggest that bcl-2 and bcl-xL are promising targets for antisense therapy of melanoma, and that the simultaneous downregulation of their expression may provide additional clinical benefit.     

Sexual behavior, human papillomavirus type 16 (HPV 16) infection, and HPV 16 seropositivity

BACKGROUND: Sexual behaviors have been linked to seropositivity for human papillomavirus (HPV) but not with the magnitude of the seroreactivity. GOALS: The objective of this analysis was to examine the association of sexual behavior, cervical HPV 16 DNA positivity at enrollment (past) and at diagnosis (current), and other potential determinants with the likelihood and magnitude of HPV 16 seropositivity at diagnosis. STUDY DESIGN: With use of stored specimens from an incidence case-control study at Kaiser Permanente (Portland, OR), women were tested for seroreactivity to HPV 16 by enzyme-linked immunosorbent assay with virus-like particles at diagnosis and were tested for past and concurrent cervical HPV 16 DNA positivity with MY09/MY11 L1 consensus primer PCR. Questionnaire data were used to ascertain past sexual behavior. RESULTS: Increased lifetime number of sex partners (P(Trend) < 0.001), past HPV 16 DNA positivity (odds ratio = 6.9; 95% confidence interval = 1.5-31), and a current cytologic diagnosis (P(Trend) < 0.03) were independently associated with HPV 16 seropositivity. Among the seropositive, only lifetime number of sex partners (P(Trend) < 0.001) and past HPV 16 DNA positivity (P = 0.003) were independently associated with mean signal strength (optical density) in an age-adjusted analysis. Women negative for past and concurrent HPV 16 DNA had a significant trend of increasing optical densities associated with greater numbers of lifetime partners (P(Trend) < 0.001). Conversely, the mean signal strength for those women who were ever HPV 16 DNA-positive during the study did not depend on lifetime numbers of sex partners (P(Trend) = 0.36). CONCLUSIONS: HPV 16 seropositivity is a surrogate for past HPV 16 infection. Circulating levels of antibodies to HPV 16 may reflect recent HPV 16 infection or the frequency of past HPV 16 infection.     

Cytotoxic activity in the serum of a patient with metastasizing nephroblastoma given intravenous infusions of alkyl-lysophospholipids in a phase I study

Summary In a phase I study a cytotoxic activity in the serum of a tumor patient given infusions of synthetic alkyl-lysophospholipid has been demonstrated. Serum samples collected after ALP infusions inhibited ³H-thymidine incorporation by human leukemic cells to an extent that correlated to the dose of ALP administered. Serum taken after the highest dose of ALP given (50 mg/kg body weight) led to complete cell destruction after 72 h in vitro. Whereas cells from the HL60 line were very sensitive to the serum cytotoxicity, K562 cells were much less affected. Cytotoxic activity was found to be cleared from the circulation in a biphasic manner; more than 50% disappeared within 6–8 h but 20–30% was still present after 4 days.Supported by DFG An 111/2     

Resistance to activated protein C (APCR) in children with venous or arterial thromboembolism

Resistance to activated protein C (APCR), in the majority of cases due to the point mutation Arg 506 Gln of the factor V gene, has emerged as the most important hereditary cause of venous thromboembolism. Using an activated thromboplastin time (aPTT) based method in the presence of APC together with a DNA technique based on the polymerase chain reaction, we investigated 37 children with venous (V: n = 19) or arterial (A: n = 18) thromboembolism and 196 age-matched healthy controls for the presence of this mutation. In the control group 10 children were detected to be heterozygous for the factor V Leiden mutation, indicating a prevalence of 5.1%. 10/19 children (52%) with venous thrombosis and 7/18 (38%) patients with arterial thromboembolism showed the common factor V gene mutation. Additional inherited coagulation disorders were found in 1/10 (V: 10%) and 2/7 (A: 28%) APC-resistant patients. Inherited coagulation disorders without APCR were diagnosed in 3/9 (V: 33%) and 2/11 (A: 18%) children. Furthermore, we diagnosed exogenous risk factors in 6/10 (V: 60%) and 2/7 (A: 28%) children with thrombosis and APCR. These data are evidence that APCR combined with exogenous reasons may play an important role in the early manifestation of thromboembolism during infancy and childhood.