HPV load and anal cytological abnormalities.

OBJECTIVES: The objective of this study was to evaluate the association between estimated human papillomavirus (HPV) viral load and abnormal cytology on anal samples. METHODS: Anal cytological samples of 42 HIV-positive patients were analysed by conventional cytology and Hybrid Capture II. RESULTS: On cytology, 30.95% (13 of 42) anal samples were positive for cytological abnormalities, 47.61% (20 of 42) were negative and 21.42% (nine of 42) were unsatisfactory. High-risk HPV infection was more frequent in anal samples with cytological abnormalities than in negative samples (P = 0.0002, Fisher's exact test), it was detected in all samples with cytological abnormalities and in 35% (seven of 20) of the negative samples. On samples with cytological abnormalities, the median of the relative light unit/cutoff (RLU/CO) value (viral load estimate) was 10.39 (1.02-572.6) and in negative samples it was 0.51 (0.26-51.70). The median of the RLU/CO value was higher in samples with cytological abnormalities when compared with the median in negative samples (P = 0.0001, Mann-Whitney U-test) and only samples with cytological abnormalities showed RLU/CO values > 100. CONCLUSIONS: The estimated high-risk HPV viral load is significantly higher in samples with cytological abnormalities than in negative anal samples and may be useful as an adjunct to anal cytology for triage of patients to high-resolution anoscopy and biopsy.

ErbB2 potentiates breast tumor proliferation through modulation of p27(Kip1)-Cdk2 complex formation: receptor overexpression does not determine growth dependency.

Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G(1) arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27(Kip1) onto Cdk2 complexes, an event preceding increased p27(Kip1) expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27(Kip1) sequestration) and the loss of p27(Kip1) from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27(Kip1) redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27(Kip1) protein levels remained constant. Antisense-mediated inhibition of p27(Kip1) expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27(Kip1) accumulation, p27(Kip1) redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G(1) block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27(Kip1) sequestration proteins, thus deregulating the G(1)/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.

Modulation of p27/Cdk2 complex formation through 4D5-mediated inhibition of HER2 receptor signaling.

The molecular mechanisms mediating the anti-proliferative effects of the murine anti-HER2 monoclonal antibody (4D5) were investigated in HER2-overexpressing human carcinoma cell lines. Treatment with 4D5 resulted in a dramatic accumulation of BT-474 breast carcinoma cells in G1; concomitant with reduced expression of proteins involved in sequestration of the cyclin E/Cdk2 inhibitor protein p27, increased association of p27 with Cdk2 complexes and Cdk2 inactivation. No equivalent effects were observed in BT-474 cells treated with a control, non-inhibitory HER2 monoclonal antibody (FRP5) or in a HER2-overexpressing cell line insensitive to 4D5 treatment (MKN7 gastric carcinoma cells), confirming the relationship between these molecular changes and 4D5-mediated inhibition of proliferation. Increased p27 expression was also observed in 4D5-treated BT-474 cells; however an antisense approach demonstrated that this increase was not required for Cdk2 inactivation or establishment of the G1 block. These data suggest that 4D5 interferes with HER2 receptor signaling, resulting in downregulation of proteins involved in p27 sequestration. This causes release of p27, allowing binding and inhibition of cyclin E/Cdk2 complexes and inhibition of G1/S progression. This model was confirmed using a second 4D5-sensitive. HER2-overexpressing breast tumor line (SKBR3), and suggests that the dependency of a given tumor cell on elevated HER2-receptor signaling for the maintenance of p27 sequestration proteins may determine the clinical response to treatment with the humanized anti-HER2 monoclonal antibody Herceptin (trastuzumab).

Molecular identification of Paracoccidioides brasiliensis by PCR amplification of ribosomal DNA.

We have amplified and sequenced the 5.8S and 28S ribosomal DNA genes and intergenic regions of Paracoccidioides brasiliensis, strain Pb01. Using primers specifically designed for both ribosomal DNA regions, we were able to discriminate between P. brasiliensis and other human pathogenic fungi by PCR. The use of this molecular marker could be important for paracoccidiodomycosis diagnosis and ecological and molecular epidemiological studies of P. brasiliensis in Latin America.