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The ABC drug transporters, including ABCG2, are well known for their ability to efflux a wide spectrum of chemotherapeutic agents, thereby conferring a multidrug-resistant phenotype. However, studies over the past several years suggest that the ABC transporters may play additional role(s) in cell survival in the face of stress inducers that are not ABCG2 substrates (i.e., nutrient deprivation, ionizing radiation, rapamycin). The mechanism by which this occurs is largely unknown. In the present study, using several cancer cell lines and their ABCG2-overexpressing sublines, we show that cells overexpressing ABCG2 were more resistant to these stressors. This resistance was associated with an elevated level of autophagy flux, as measured by a higher rate of SQSTM1/p62 degradation and greater accumulation of LC3-II when compared to parental cells. Knockdown of ABCG2 reduced autophagic activity in resistant cells to a level similar to that observed in parental cells, confirming that the enhanced autophagy was ABCG2-dependent. Moreover, using cell viability, apoptosis, and clonogenic assays, we demonstrated that the ABCG2-expressing cells were more resistant to amino acid starvation and radiation-induced cell death. Importantly, knockdown of the critical autophagy factors ATG5 and ATG7 greatly reduced cell survival, verifying that enhanced autophagy was critical for this effect. Taken together, these data indicate that autophagy induced by various stressors is enhanced/accelerated in the presence of ABCG2, resulting in delayed cell death and enhanced cell survival. This defines a new role for this transporter, one with potential clinical significance.

E2F-1, a key transcription factor necessary for cell growth, DNA repair, and differentiation, is an attractive target for development of anticancer drugs in tumors that are E2F "oncogene addicted". We identified a peptide isolated from phage clones that bound tightly to the E2F-1 promoter consensus sequence. The peptide was coupled to penetratin to enhance cellular uptake. Modeling of the penetratin-peptide (PEP) binding to the DNA E2F-1 promoter demonstrated favorable interactions that also involved the participation of most of the penetratin sequence. The penetratin-peptide (PEP) demonstrated potent in vitro cytotoxic effects against a range of cancer cell lines, particularly against Burkitt lymphoma cells and small cell lung cancer (SCLC) cells. Further studies in the H-69 SCLC cell line showed that the PEP inhibited transcription of E2F-1 and also several important E2F-regulated enzymes involved in DNA synthesis, namely, thymidylate synthase, thymidine kinase, and ribonucleotide reductase. As the PEP was found to be relatively unstable in serum, it was encapsulated in PEGylated liposomes for in vivo studies. Treatment of mice bearing the human small cell lung carcinoma H-69 with the PEP encapsulated in PEGylated liposomes (PL-PEP) caused tumor regression without significant toxicity. The liposome encapsulated PEP has promise as an antitumor agent, alone or in combination with inhibitors of DNA synthesis.

Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

Alternative splicing of pre-mRNA contributes significantly to human proteomic complexity, playing a key role in development, gene expression and, when aberrant, human disease onset. Many of the factors involved in alternative splicing have been identified, but little is known about their regulation. Here we report that caffeine regulates alternative splicing of a subset of cancer-associated genes, including the tumor suppressor KLF6. This regulation is at the level of splice site selection, occurs rapidly and reversibly, and is concentration dependent. We have recapitulated caffeine-induced alternative splicing of KLF6 using a cell-based minigene assay and identified a "caffeine response element" within the KLF6 intronic sequence. Significantly, a chimeric minigene splicing assay demonstrated that this caffeine response element is functional in a heterologous context; similar elements exist within close proximity to caffeine-regulated exons of other genes in the subset. Furthermore, the SR splicing factor, SC35, was shown to be required for induction of the alternatively spliced KLF6 transcript. Importantly, SC35 is markedly induced by caffeine, and overexpression of SC35 is sufficient to mimic the effect of caffeine on KLF6 alternative splicing. Taken together, our data implicate SC35 as a key player in caffeine-mediated splicing regulation. This novel effect of caffeine provides a valuable tool for dissecting the regulation of alternative splicing of a large gene subset and may have implications with respect to splice variants associated with disease states.

Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively. However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved. In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoprotein-encoding gene, MDR1. Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level of MDR1 mRNA 20-fold. Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10- to 15-fold induction of promoter activity. Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation. Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation. Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1 promoter by TSA. Moreover, NF-YA and P/CAF were shown to interact in vitro. This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified.

Nucleotide sequence analysis of two cDNA clones, one shown to direct the synthesis in Escherichia coli of the pI 6.7 form of the 20,000-molecular-weight class of Chinese hamster lung cell dihydrofolate reductase, and the other shown to direct the synthesis of the pI 6.5 form of the 21,000-molecular-weight class of the enzyme, has revealed the following: (i) the differences in physical and enzymatic properties displayed by these two proteins are due to two variations in their respective amino acid sequences with the conversion of Leu to Phe at position 22 probably responsible for the differential sensitivity of these two enzymes to methotrexate and methasquin; (ii) the multiple mRNAs responsible for the synthesis of each of these proteins differ in size due, at least in part, to a length heterogeneity at their 3' ends; (iii) these two proteins are encoded by different genes; and (iv) the sequence AAATATA appears to be a major polyadenylation signal in one Chinese hamster lung cell dihydrofolate reductase gene and a minor signal in another.