Naive CD4+ T Cells Harbor a Large Inducible Reservoir of Latent, Replication-competent Human Immunodeficiency Virus Type 1.

BACKGROUND: The latent human immunodeficiency virus type 1 (HIV-1) reservoir represents a major barrier to a cure. Based on the levels of HIV-1 DNA in naive (TN) vs resting memory CD4+ T cells, it is widely hypothesized that this reservoir resides primarily within memory cells. Here, we compared virus production from TN and central memory (TCM) CD4+ T cells isolated from HIV-1-infected individuals on suppressive therapy. METHODS: CD4+ TN and TCM cells were purified from the blood of 7 HIV-1-infected individuals. We quantified total HIV-1 DNA in the CD4+ TN and TCM cells. Extracellular virion-associated HIV-1 RNA or viral outgrowth assays were used to assess latency reversal following treatment with anti-CD3/CD28 monoclonal antibodies (mAbs), phytohaemagglutinin/interleukin-2, phorbol 12-myristate 13-acetate/ionomycin, prostratin, panobinostat, or romidepsin. RESULTS: HIV-1 DNA was significantly higher in TCM compared to TN cells (2179 vs 684 copies/106 cells, respectively). Following exposure to anti-CD3/CD28 mAbs, virion-associated HIV-1 RNA levels were similar between TCM and TN cells (15 135 vs 18 290 copies/mL, respectively). In 4/7 donors, virus production was higher for TN cells independent of the latency reversing agent used. Replication-competent virus was recovered from both TN and TCM cells. CONCLUSIONS: Although the frequency of HIV-1 infection is lower in TN compared to TCM cells, as much virus is produced from the TN population after latency reversal. This finding suggests that quantifying HIV-1 DNA alone may not predict the size of the inducible latent reservoir and that TN cells may be an important reservoir of latent HIV-1.

Recruitment of NCOR1 to VDR target genes is enhanced in prostate cancer cells and associates with altered DNA methylation patterns.

The current study investigated transcriptional distortion in prostate cancer cells using the vitamin D receptor (VDR) as a tool to examine how epigenetic events driven by corepressor binding and CpG methylation lead to aberrant gene expression. These relationships were investigated in the non-malignant RWPE-1 cells that were 1α,25(OH)(2)D(3) responsive (RWPE-1) and malignant cell lines that were 1α,25(OH)(2)D(3) partially responsive (RWPE-2) and resistant (PC-3). These studies revealed that selective attenuation and repression of VDR transcriptional responses in the cancer cell lines reflected their loss of antiproliferative sensitivity. This was evident in VDR target genes including VDR, CDKN1A (encodes p21( (waf1/cip1) )) and GADD45A; NCOR1 knockdown alleviated this malignant transrepression. ChIP assays in RWPE-1 and PC-3 cells revealed that transrepression of CDKN1A was associated with increased NCOR1 enrichment in response to 1α,25(OH)(2)D(3) treatment. These findings supported the concept that retained and increased NCOR1 binding, associated with loss of H3K9ac and increased H3K9me2, may act as a beacon for the initiation and recruitment of DNA methylation. Overexpressed histone methyltransferases (KMTs) were detectable in a wide panel of prostate cancer cell lines compared with RWPE-1 and suggested that generation of H3K9me2 states would be favored. Cotreatment of cells with the KMT inhibitor, chaetocin, increased 1α,25(OH)(2)D(3)-mediated induction of CDKN1A expression supporting a role for this event to disrupt CDKN1A regulation. Parallel surveys in PC-3 cells of CpG methylation around the VDR binding regions on CDKN1A revealed altered basal and VDR-regulated DNA methylation patterns that overlapped with VDR-induced recruitment of NCOR1 and gene transrepression. Taken together, these findings suggest that sustained corepressor interactions with nuclear-resident transcription factors may inappropriately transform transient-repressive histone states into more stable and repressive DNA methylation events.

Fenugreek: a naturally occurring edible spice as an anticancer agent.

In recent years, various dietary components that can potentially be used for the prevention and treatment of cancer have been identified. In this study, we demonstrate that extract (FE) from the seeds of the plant Trigonella foenum graecum, commonly called fenugreek, are cytotoxic in vitro to a panel of cancer but not normal cells. Treatment with 10-15 ug/mL of FE for 72 h was growth inhibitory to breast, pancreatic and prostate cancer cell lines (PCa). When tested at higher doses (15-20 ug/mL), FE continued to be growth inhibitory to PCa cell lines but not to either primary prostate or hTert-immortalized prostate cells. At least part of the growth inhibition is due to induction of cell death, as seen by incorporation of Ethidium Bromide III into cancer cells exposed to FE. Molecular changes induced in PCa cells are: in DU-145 cells: downregulation of mutant p53, and in PC-3 cells upregulation of p21 and inhibition of TGFbeta induced phosphorylation of Akt. The surprising finding of our studies is that death of cancer cells occurs despite growth stimulatory pathways being simultaneously upregulated (phosphorylated) by FE. Thus, these studies add another biologically active agent to our armamentarium of naturally occurring agents with therapeutic potential.

In vitro and in vivo effects of the conformationally restricted polyamine analogue CGC-11047 on small cell and non-small cell lung cancer cells.

PURPOSE: Polyamines are essential for normal growth; however, the requirement for, and the metabolism of, polyamines are frequently dysregulated in cancer. Polyamine analogues have demonstrated promising preclinical results in multiple model systems of cancer, but their clinical utility has been limited by apparent toxicity. A representative compound of a new generation of short chain, conformationally restricted polyamine analogues, CGC-11047 has been synthesized and ongoing phase I clinical trials indicate it to be well tolerated at weekly doses of 610 mg (dose escalation is still in progress). Therefore, studies were designed to gain a better understanding of its effects on cellular polyamine biochemistry and efficacy in the treatment of human lung cancer models in vitro and in vivo. METHODS: Human lung cancers cell lines representing non-small cell and small cell lung cancers were investigated for their growth and biochemical response to CGC-11047. Effects of in vitro treatment with CGC-11047 on cell growth, the activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC), and the expression and activity of the polyamine catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxides (SMO) were measured. Additionally, the overall effects on intracellular polyamine pools were monitored. Finally, the in vivo efficacy of CGC-11047 in the treatment of a nude mouse model of human non-small cell lung cancer was evaluated. RESULTS: CGC-11047 effectively inhibited the growth of both small cell and non-small cell lung cancer cells in vitro. The greatest biochemical effects were observed in the non-small cell lung cancer cells where in addition to a profound down regulation of ODC activity, there was a significant increase in polyamine catabolism leading to a greater degree of polyamine pool depletion and greater accumulation of CGC-11047 when compared with the changes observed for the small cell lines. Importantly, CGC-11047 was found to be highly significant (P < 0.0001) in delaying the progression of established tumors in an in vivo model of human non-small cell lung cancer. CONCLUSION: CGC-11047 represents a promising new polyamine analogue that warrants further preclinical and, potentially, clinical evaluation in lung cancer.