Slow Acceptance of Universal Antiretroviral Therapy (ART) Among Mothers Enrolled in IMPAACT PROMISE Studies Across the Globe.

The PROMISE trial enrolled asymptomatic HIV-infected pregnant and postpartum women not eligible for antiretroviral treatment (ART) per local guidelines and randomly assigned proven antiretroviral strategies to assess relative efficacy for perinatal prevention plus maternal/infant safety and maternal health. The START study subsequently demonstrated clear benefit in initiating ART regardless of CD4 count. Active PROMISE participants were informed of results and women not receiving ART were strongly recommended to immediately initiate treatment to optimize their own health. We recorded their decision and the primary reason given for accepting or rejecting the universal ART offer after receiving the START information. One-third of participants did not initiate ART after the initial session, wanting more time to consider. Six sessions were required to attain 95% uptake. The slow uptake of universal ART highlights the need to prepare individuals and sensitize communities regarding the personal and population benefits of the "Treat All" strategy.

Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity.

We have recently reported that treatment with gemcitabine, a potent chemotherapeutic agent and radiation sensitizer, stimulates phosphorylation of the epidermal growth factor receptor (EGFR). Because phosphorylation of EGFR is known to precede receptor degradation, we hypothesized that gemcitabine treatment may also result in EGFR degradation. In two human head and neck cancer cell lines, UMSCC-1 and UMSCC-6, we demonstrated an approximately 80% decrease in total EGFR levels at 72 h after a 2-h treatment with 1 muM gemcitabine. Neither cisplatin nor 5-fluorouracil, which are used to treat head and neck cancer, caused EGFR degradation. EGFR downregulation did not occur at the level of transcription, as assessed by reverse transcription-polymerase chain reaction (RT-PCR), but instead occurred via phosphorylation and ubiquitination of the receptor along a proteosome/lysosome-mediated pathway. Inhibition of EGFR degradation, by either pretreatment with the EGFR tyrosine kinase inhibitor gefitinib or by exposure to the proteosome/lysosome inhibitor MG132, significantly reduced gemcitabine-induced cell death. These results suggest that EGFR degradation may be a novel mechanism for gemcitabine-mediated cell death. These findings also indicate that caution should be exercised when combining gemcitabine with agents that may prevent EGFR degradation, such as EGFR tyrosine kinase inhibitors administered in a suboptimal sequence or proteosome inhibitors.

The potential of 5-fluorocytosine/cytosine deaminase enzyme prodrug gene therapy in an intrahepatic colon cancer model.

Colorectal cancer can metastasize to the liver, but remain liver confined for years. A critical step in developing treatments for intrahepatic cancer involves assessment in an orthotopic intrahepatic model. The purpose of this study was to develop a noninvasive intrahepatic tumor model to study the efficacy of 5-flucytosine/yeast cytosine deaminase (5FC/yCD)-based gene therapy for liver tumors. Luciferase expressing human colorectal carcinoma (HT-29luc) cells were generated by retroviral infection and implanted in the left liver lobe of nude mice. The bioluminescence was measured every week for a period of 1 month, then animals were killed and tumors were measured by calipers. After we found a correlation between photon counts and tumor size, animals were implanted with tumors composed of either 0%, 10%, or 100% yCD/HT-29luc cells, and treated with 5FC. Tumor bioluminescence was measured during treatment and tumor histology examined at the time of death. We found that 5FC caused significant regression of yCD expressing tumors. Furthermore, visible tumors at the time of death, which emitted little bioluminescence, contained little or no viable tumor. We then developed an adenoviral vector for yCD. Intraperitoneal administration of adenovirus containing yCD led to the production of yCD enzyme within intrahepatic tumors. These results suggest that (1) intrahepatic cancer responds to 5FC when cells express yCD; (2) the luciferin-luciferase system permits non-invasive real time imaging of viable intrahepatic cancer; and (3) this system can be used to carry out gene therapy experiments using yCD adenovirus.

Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins.

The advent of DNA microarray technology will likely have a major impact on the molecular classification and understanding of human cancer. Obtaining a global perspective of proteins expressed in cancer cells is considerably more challenging. Here we describe a microarray-based platform that can be used to measure protein levels and activities in a complex biological milieu such as a cellular lysate. Using a protein microarray made up of 1920 elements (146 distinct antibodies) we were able to monitor alterations of protein levels in LoVo colon carcinoma cells treated with ionizing radiation. The protein microarray approach revealed radiation-induced up-regulation of apoptotic regulators including p53, DNA fragmentation factor 40/caspase activated DNase, DNA fragmentation factor 45/inhibitor of caspase activated DNase, tumor necrosis factor-related apoptosis-inducing ligand, death receptor 5, decoy receptor 2, FLICE-like inhibitory protein, signal transducers and activators of transcription 1alpha, and uncoupling protein 2, among others. Consistent with this observation, an increased percentage of apoptosis was observed in irradiated LoVo cells. Interestingly, we also observed radiation-induced down-regulation of carcinoembryonic antigen, a prototypic cancer biomarker. Selected proteins assessed by microarray were validated by traditional immunoblotting. Taken together, our work suggests that protein/antibody microarrays will facilitate high-throughput proteomic studies of human cancer and carcinogenesis.

Yeast cytosine deaminase improves radiosensitization and bystander effect by 5-fluorocytosine of human colorectal cancer xenografts.

The efficacy of cancer gene therapy using bacterial cytosine deaminase (bCD)/5-fluorocytosine (5-FC) enzyme/prodrug strategy is limited by the inefficiency of cytosine deaminase (CD)-catalyzed conversion of 5-FC into 5-fluorouracil (5-FU). We have shown previously that yeast CD (yCD) is more efficient at the conversion of 5-FC than bCD. In the current study, we hypothesized that the increased production of 5-FU by yCD would enhance the efficacy of the CD/5-FC treatment strategy by increasing the bystander effect as well as the efficacy of radiotherapy because of the radiosensitizing capacity of 5-FU. To test this hypothesis, we generated stable HT29 human colon cancer cell lines expressing either bCD (HT29/bCD) or yCD (HT29/yCD). The amount of 5-FU produced in HT29/yCD tumors after a single injection of 5-FC (1000 mg/kg, i.p.) was 15-fold higher than that produced in HT29/bCD tumors. In tumor-bearing nude mice, the average minimum relative tumor size (compared with pretreatment values) of HT29/bCD tumors treated with 5-FC and radiation (500 mg/kg i.p. and 3 Gy, 5 days a week for 2 weeks) was 0.55+/-0.1, compared with 0.01+/-0.01 in HT29/yCD tumors (P = 0.002). Moreover, an increased cytotoxic and radiosensitizing effect of 5-FC on bystander cells was observed in vitro and in vivo when yCD was expressed in HT29 cells instead of bCD. In mice bearing HT29 tumors containing 10% HT29/yCD cells, the combined treatment resulted in a minimum tumor size of 0.20+/-0.07 compared with 0.60+/-0.1 in 10% HT29/bCD cells (P < 0.001). These results demonstrate that the use of yCD in the CD/5-FC strategy has a high potential to improve the therapeutic outcome of combined gene therapy and radiotherapy in cancer patients.

Development of differential cytotoxic compounds containing nitrosourea and benzothiazine nucleus.

The in vivo activity of 4 new benzothiazinyl-nitrosourea compounds was investigated against Sarcoma-180 (S-180) and Ehrlich Ascitic Carcinoma (EAC) induced ascitic and solid tumors. EAC solid tumor was found to be the most sensitive, where one compound (no 4) inhibited tumor growth to only 3 per cent of the control value. All the 4 compounds tested were found to be toxicologically more selective than 5-fluorouracil and 6-mercaptopurine drugs. The reason for this selective toxicity may be attributed to the inhibition of isocyanate moiety in these compounds which causes toxicity to normal cells via a carbamoylation reaction. However, they may still remain potent, since they decompose into an alkylating carbonium species and a charge transfer complex which may interact with DNA via alkylation and intercalation reactions, respectively.

Evaluation of certain ureas and nitrosoureas of 2,3-dihydro-1,4-benzothiazines against Sarcoma-180 solid tumors in vivo.

Different derivatives of nitrosoureas were synthesized by a long sequence of reactions and were evaluated for their anticancer activities against female Swiss albino mice, 6-8 weeks old, weighing 18-24 g and bearing Sarcoma-180 (S-180) ascitic tumor. Experimental protocols include injecting a total of 2 x 10(5) S-180 viable ascitic cells s.c. on day 0 in the left flank of mice to obtain solid tumors, followed by the treatment of urea/nitrosourea derivatives on days 1, 5 and 9 at different dose levels. Of the 32 compounds tested, eight were found highly active against solid tumors with significant growth delay and tumor weight inhibition ratio. 5-Fluorouracil was injected s.c. as referent compound to positive control animals.