A phase I study of decitabine with pegylated interferon α-2b in advanced melanoma: impact on DNA methylation and lymphocyte populations.

BACKGROUND: Melanoma cell lines treated with decitabine show upregulation of cancer antigens, and interferon-α upregulates MHC Class I antigens in cancer cells, leading to enhanced T-cell recognition and T-cell mediated tumor apoptosis. We evaluated the synergy between the hypomethylating effects of decitabine and the immunomodulatory effects of interferon in a combination regimen administered to advanced melanoma patients in a phase 1 trial. METHODS: Patients with one prior systemic therapy were eligible. Using a modified 3 + 3 design, patients received escalating doses of decitabine and pegylated interferon α-2b (PEG-IFN) during every 28-day treatment cycle. Global DNA methylation was measured on days 1 and 5 of cycles 1 and 3. Cytokine profiling and quantification of T-cell subpopulations by FACS were performed at baseline and cycle 3. RESULTS: Seventeen patients were assigned to one of four dose levels. Decitabine 15 mg/m2/d + PEG-IFN 3 µg/kg was the maximum tolerated dose (MTD). Grade 3/4 cytopenias were seen across all dose levels: anemia (1), neutropenia (7), and thrombocytopenia (2). One patient remained progression-free for 37 weeks. The other 16 patients progressed at or before 12 weeks. Median overall survival was 39 weeks. Hypomethylation was seen at all dose levels. Due to treatment-induced lymphocytopenia, absolute changes in T-cell populations post-treatment were too small to be meaningfully interpreted. CONCLUSIONS: The response to this combination regimen was characterized by significant myelosuppression, particularly neutropenia. Although disappointing efficacy and slow accrual led to early closure of the trial, hypomethylation showed pharmacodynamic evidence of a therapeutic effect of decitabine at all dose levels.

Regulation of alphaA-crystallin gene expression. Lens specificity achieved through the differential placement of similar transcriptional control elements in mouse and chicken.

The lens-preferred mouse alphaA-crystallin gene contains a conserved stretch (proximal element 2, +24/+43) in its 5'-noncoding region that we have previously shown binds nuclear proteins of lens and non-lens cells. The 5'-half of this sequence (PE2A, +25/+32) has consensus binding sites for AP-1 and other transcription factors. We show here by deletion experiments that PE2A is important for activity of the mouse alphaA-crystallin promoter and mediates phorbol ester and c-Jun responsiveness of this promoter in transfected lens cells. In vitro protein binding studies suggest that AP-1 complexes are capable of binding to PE2A. Our findings suggest that PE2A plays a role in mouse alphaA-crystallin gene expression through AP-1-mediated regulatory mechanisms. We propose that the mouse and chicken alphaA-crystallin genes are expressed with lens specificity using a similar assortment of transcription factors but with a different physical arrangement of their respective cis-elements within the promoter region. A fundamental role for AP-1 in lens-preferred expression of crystallin genes is consistent with the idea that a redox-sensitive mechanism is a selective force for recruiting lens crystallins.

Lens-specific activity of the mouse alpha A-crystallin promoter in the absence of a TATA box: functional and protein binding analysis of the mouse alpha A-crystallin PE1 region.

Lens-specific expression of the mouse alpha A-crystallin gene is regulated at the level of transcription. Here, we have studied the role of the PE1 region, which contains the TATA box (-31/-26) and the immediately adjacent PE1B sequence (-25/-12), in transcriptional regulation. Deletions within either the TATA box or PE1B sequence eliminated promoter activity in transfected lens cells. Surprisingly, these deletions did not eliminate lens-specific promoter activity of the transgene of transgenic mice. Transcription of the transgene with a TATA-deleted promoter initiated at multiple sites in the lenses of the transgenic mice. Footprint analysis revealed that the entire PE1 region was protected by nuclear extracts prepared from lens cells which express the alpha A-crystallin gene and from fibroblasts which do not express the gene. The -37/+3 region formed three specific EMSA complexes using lens cell nuclear extracts, while a similar but much less intense pattern was observed when a fibroblast nuclear extract was used. Competition experiments indicated that these complexes were not due to the binding of TBP to the TATA box, but rather to the binding of other nuclear proteins to the PE1B -25/-19 region. A series of co-transfection competition studies in vivo also suggested the functional importance of proteins binding in the -25/-19 region. The PE1B protein-DNA interactions appear to be conserved in the chicken, rodent and human alpha A-crystallin gene as well as within the alpha A- and alpha B-crystallin genes in the mouse. Our findings indicate that the PE1B region is important for mouse alpha A-crystallin promoter activity; the proximity of this site to the TATA box raises the possibility for cooperativity or competition between TBP and PE1B-bound proteins.

Functional analysis of chicken vimentin distal promoter regions in cultured lens cells.

Synthesis of the cytoskeletal intermediate filament protein vimentin (Vim) in the lens is unexpected due to the mesenchymal preference of Vim-encoding gene (Vim) expression and the epithelial origin of the lens. Previous studies indicated that chicken Vim gene expression in cultured lens cells is regulated by both positive- and negative-acting sequence elements within the first -767 nucleotides (nt) of its promoter. Here, we demonstrate the existence of additional upstream chicken Vim promoter elements which function in transfected lens cells. Sequences within the nt -1360/-1156 region repressed promoter activity in transfected lens cells to levels lower than that observed for the previously defined more proximal repressor elements. The -1612/-1360 region activated promoter activity to levels similar to those observed for the strongest previously defined proximal promoter. The nt sequence analysis of the upstream promoter region revealed the presence of multiple consensus repressor and activator transcription-factor-binding sites. Several of these sites have been implicated for lens expression of enzyme-crystallin-encoding genes (cry), suggesting that Vim expression may share features with the cry genes for recruitment and high-level expression in the lens.

Functional redundancy of the DE-1 and alpha A-CRYBP1 regulatory sites of the mouse alpha A-crystallin promoter.

Previous studies have implicated the DE-1 (-111/-106) and alpha A-CRYBP1 (-66/-57) sites for activity of the mouse alpha A-crystallin promoter in transiently transfected lens cells. Here we have used the bacterial chloramphenicol acetyltransferase (CAT) reporter gene to test the functional importance of the putative DE-1 and alpha A-CRYBP1 regulatory elements by site-specific and deletion mutagenesis in stably transformed alpha TN4-1 lens cells and in transgenic mice. FVB/N and C57BL/6 x SJL F2 hybrid transgenic mice were assayed for CAT activity in the lens, heart, lung, kidney, spleen, liver, cerebrum, and muscle. F0, F1, and F2 mice from multiple lines carrying single mutations of the DE-1 or alpha A-CRYBP1 sites showed high levels of CAT activity in the lens, but not in any of the non-lens tissues. By contrast, despite activity of the wild-type promoter, none of the mutant promoter/CAT constructs were active in the transiently transfected and stably transformed lens cells. The mice carrying transgenes with either site-specific mutations in both the DE-1 and alpha A-CRYBP1 sites or a deletion of the entire DE-1 and part of the alpha A-CRYBP1 site (-60/+46) fused to the CAT gene did not exhibit CAT activity above background in any of the tissues examined, including the lens. Our results thus indicate that the DE-1 and alpha A-CRYBP1 sites are functionally redundant in transgenic mice. Moreover, the present data coupled with previous transfection and transgenic mouse experiments suggest that this functional redundancy is confined to lens expression within the mouse and is not evident in transiently transfected and stably transformed lens cells, making the cultured lens cells sensitive indicators of functional elements of crystallin genes.

Estimation of body composition changes during weight cycling by bioelectrical impedance analysis in rats.

The effects of body weight cycling (WC) in rats on body composition (BC) and feeding efficiency were studied. The usefulness of estimating BC by bioelectrical impedance analysis (BIA) was also examined. Female Sprague-Dawley rats were divided into high-fat ad libitum feeding, either noncycling or cycling, or restricted feeding (75% of control feed) cycling groups. Control rats were fed a regular laboratory ad libitum diet and did not cycle. All rats were killed at the end of week 61. A BIA unit was used at each stage of WC to obtain resistance and reactance readings. Final BC was determined by chemical analysis. On the basis of the final chemical analysis and BIA measurements, an equation was established and applied to estimate BC at each stage of WC: fat-free mass (g) = 0.38 x body wt (g) + 13.8 x [length (cm)2/resistance] + 70.9 (r = 0.95, P < 0.001). High-fat ad libitum feeding induced rapid body weight and fat gains as well as an elevated feeding efficiency and an internal fat-to-subcutaneous fat ratio, regardless of whether the rats cycled. This change in fat mass was clearly detected by the BIA. Although rats fed restricted diets had similar body weights as did control rats, they had a significantly higher internal fat-to-subcutaneous fat ratio. Thus, not only the amount of food but also the composition of the diet is important for proper weight management. The BIA method is capable of detecting the body fat mass change during WC.

Long-term exercise training and retirement in genetically obese rats: effects on food intake, feeding efficiency and carcass composition.

Short-term physical exercise (EX) can reduce body weight and fat gain in obese humans and animals. However, the beneficial effects of physical exercise are not long-lasting. In this study, the effects of long-term physical exercise and retirement from exercise (R) on body weight, body composition and fat distribution were examined in genetically obese (OB) and lean (LE) female rats. Fifty OB and 45 LE rats, four weeks old, were divided into EX (swimming, 2h/day, 5 days/week) or sedentary (SD) groups. At the end of the 28th week of treatment, EX groups were further divided into continued EX or R groups for another 11-12 weeks. It was found that at the end of the 28th week EX had reduced the rate of weight gain in OB and LE rats. Percentage body fat was only reduced in OBEX rats and this was achieved by a significant reduction of subcutaneous fat mass. At the end of the 40th week, EX had further reduced the weight gain, fat mass and body fat percentage in OBEX rats while only body fat percentage was reduced in the LEEX group. Retirement from exercise reversed these phenomena. Thus there were no differences between OBSD and OBEX-R rats in body weight, fat mass and percentage body fat. However, the OBEX-R group had a significantly higher amount of internal fat than the other two OB groups. Therefore, exercise, even long-term to cover the entire fat cell proliferation period, still only exerted temporary beneficial effects in OB rats. After retirement, the beneficial effects all disappeared rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)