E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis, To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes, Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes, Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas.

Association between HIV-1 infection, the etiology of genital ulcer disease, and response to syndromic management

Background: Reports on the effect of HIV-1 infection on healing rates of ulcers are conflicting. Goal: The goal was to determine the etiology and response to treatment of genital ulcer disease (GUD) in relation to HIV-1 infection. Study Design: This was a cohort study of patients with GUD treated with local syndromic management protocols. Results: Among the 587 recruited, the prevalences of infections due to HSV, Treponema pallidum, Chlamydia trachomatis (lymphogranuloma venereum [LGV]), Haemophilus ducreyi, Calymmatobacterium granulomatis, and HIV-1 were 48%, 14%, 11%, 10%, 1%, and 75%, respectively. The prevalence of T pallidum was higher among men (P = 0.03), and an association was seen among HIV-1-seronegatives on univariate and multivariate analyses (P < 0.001; P = 0.01). The prevalence of C trachomatis (LGV) was higher among females (P = 0.004), and an association was seen among HIV-1-seropositives on univariate analysis (P = 0.04). At follow-up, 40/407 (10%) showed a decreased healing tendency, not associated with ulcer etiology or HIV-1 seropositivity. Conclusion: Response to syndromic management of GUD was acceptable and not associated with HIV-1 coinfection.

Suppression of keratinocyte growth and differentiation by transforming growth factor beta 1 involves multiple signaling pathways

Transforming growth factor beta1 treatment of keratinocytes results in a suppression of differentiation, an induction of extracellular matrix production, and a suppression of growth. In this study we utilized markers specific for each of these functions to explore the signaling pathways involved in mediating these transforming-growth-factor-beta1-induced activities. In the first instance, we found that the induction of extracellular matrix production (characterized by 3TP-Lux reporter activity) was induced in both keratinocytes and a keratinocyte-derived carcinoma cell line, SCC25, in a dose-dependent manner. Furthermore, transforming growth factor beta1 also suppressed the differentiation-specific marker gene, transglutaminase type 1, in both keratinocytes and SCC25 cells. In contrast, transforming growth factor beta1 inhibited proliferation of keratinocytes but did not cause growth inhibition in the SCC25 cells. Transforming-growth-factor-beta1-induced growth inhibition of keratinocytes was characterized by decreases in DNA synthesis, accumulation of hypophosphorylated Rb, and the inhibition of the E2F:Rb-responsive promoter, cdc2, and an induction of the p21 promoter. When the negative regulator of transforming growth factor beta1 signaling, SMAD7, was overexpressed in keratinocytes it could prevent transforming-growth-factor-beta1-induced activation of the 3TP-Lux and the p21 promoter. SMAD7 could also prevent the suppression of the transglutaminase type 1 by transforming growth factor beta1 but it could not inhibit the repression of the cdc2 promoter. These data indicate that the induction of 3TP-Lux and p21 and the suppression of transglutaminase type 1 are mediated by a different proximate signaling pathway to that regulating the suppression of the cdc2 gene. Combined, these data indicate that the regulation of transforming growth factor beta1 actions are complex and involve multiple signaling pathways.

Design of a polyepitope construct for the induction of HLA-A0201-restricted HIV 1-specific CTL responses using HLA-A*0201 transgenic, H-2 class IKO mice

HLA-A*0201 transgenic, H-2D(b)/mouse beta2-microglobulin double-knockout mice were used to compare and optimize the immunogenic potential of 17HIV 1-derived, HLA-A0201-restricted epitopic peptides. A tyrosine substitution in position 1 of the epitopic peptides, which increases both their affinity for and their HLA-A0201 molecule stabilizing capacity, was introduced in a significant proportion, having verified that such modifications enhance their immunogenicity in respect of their natural antigenicity. Based on these results, a 13-polyepitope construct was inserted in the pre-S2 segment of the hepatitis B middle glycoprotein and used for DNA immunization. Long-lasting CTL responses against most of the inserted epitopes could be elicited simultaneously in a single animal with cross-recognition in several cases of their most common natural variants.

Mechanistic aspects of HIV-1 reverse transcription initiation

During reverse transcription, the positive-strand HIV-1 RNA genome is converted into a double-stranded DNA copy which can be permanently integrated into the host cell genome. Recent analyses show that HIV-1 reverse transcription is a highly regulated process. The initiation reaction can be distinguished from a subsequent elongation reaction carried out by a reverse transcription complex composed of (at least) heterodimeric reverse transcriptase, cellular tRNA(lys3) and HIV-1 genomic RNA sequences. In addition, viral factors including Tat, Nef, Vif, Vpr, IN and NCp7, cellular proteins, and TAR RNA and other RNA stem-loop structures appear to influence this complex and contribute to the efficiency of the initiation reaction. As viral resistance to many antiretroviral compounds is a continuing problem, understanding the ways in which these factors influence the reverse transcription complex will likely lead to novel antiretroviral strategies. Copyright (C) 2001 John Wiley Sons, Ltd.

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid D-Phe in aSvg-30 [D-Phe11,His12] Svg((11-40)) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl) benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[ His12] Svg was tested for its ability to displace [I-125-Tyr0] oCRF or [I-125-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 ( rCRF(1)) or mouse CRF receptor, type 2beta (mCRF(2beta)). Furthermore, the ability of ATB- [His12] Svg((12-40)) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF(1) (HEK-rCRF(1) cells) or mCRF(2beta) (HEK-mCRF(2beta) cells) was determined. Unlike astressin and photo astressin, ATB- [His12]Svg((12-40)) showed high selective binding to mCRF(2beta) (K-i = 3.1 +/- 0.2 nM) but not the rCRF(1) receptor (K-i = 142. 5 +/- 22.3 nM) and decreased Svg-stimulated cAMP activity in mCRF(2beta)-expressing cells in a similar fashion as aSvg-30. A66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB- [His12]Svg((12-40)) was covalently linked to mCRF(2beta) receptor. The specificity of the photoactivatable I-125-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).

The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions

Background: In the presence of dNTPs, intact HIV-1 virions are capable of reverse transcribing at least part of their genome, a process known as natural endogenous reverse transcription (NERT). PCR analysis of virion DNA produced by NERT revealed that the first strand transfer reaction (1stST) was inefficient in intact virions, with minus strand (-) strong stop DNA (ssDNA) copy numbers up to 200 times higher than post-1stST products measured using primers in U3 and U5. This was in marked contrast to the efficiency of 1stST observed in single-round cell infection assays, in which (-) ssDNA and U3-U5 copy numbers were indistinguishable. Objectives: To investigate the reasons for the discrepancy in first strand transfer efficiency between intact cell-free virus and the infection process. Study design: Alterations of both NERT reactions and the conditions of cell infection were used to test whether uncoating and/or entry play a role in the discrepancy in first strand transfer efficiency. Results and Conclusions: The difference in 1stST efficiency could not be attributed simply to viral uncoating, since addition of very low concentrations of detergent to NERT reactions removed the viral envelope without disrupting the reverse transcription complex, and these conditions resulted in no improvement in 1stST efficiency. Virus pseudotyped with surface glycoproteins from either vesicular stomatitis virus or amphotrophic murine leukaemia virus also showed low levels of 1stST in low detergent NERT assays and equivalent levels of (-) ssDNA and 1stST in single-round infections of cells, demonstrating that the gp120-mediated infection process did not select for virions capable of carrying out 1stST. These data indicate that a post-entry event or factor may be involved in efficient HIV-1 reverse transcription in vivo. (C) 2002 Elsevier Science B.V. All rights reserved.