The p53 tumor suppressor protein induces apoptosis in response to genotoxic and environmental stresses. Recent studies have revealed the existence of a transcriptionindependent mitochondrial p53 apoptotic pathway; however, the mechanism that regulates its translocation to the mitochondria has been unknown. In this study, we show that the tumor suppressor Tid1 forms a complex with p53 under hypoxic conditions that directs p53 translocation to the mitochondria and the subsequent initiation of the mitochondrial apoptosis pathway. Loss of Tid1 expression abrogated p53 translocation to the mitochondria and inhibited apoptosis, whereas the over-expression of Tid1 promoted p53 mitochondrial localization and apoptosis. Tid1's mitochondrial signal sequence and DnaJ domain were both required for the movement of the p53-Tid1 complex from the cytosol to the mitochondria. When Tid is over-expressed in cancer cell lines expressing mutant p53 isoforms defective in transcriptional activity, mitochondrial localization and pro-apoptotic activities of the mutant p53 proteins was restored. Our results establish Tid1 as a novel regulator of p53-mediated apoptosis, and suggest that therapies designed to enhance Tid1's function in promoting mitochondrial localization of p53 and apoptosis could be an effective therapy in many cancers.

Nucleoside transporters in kidney mediate renal reabsorption and secretion of nucleosides. Using RT-PCR, we demonstrated mRNAs encoding hENT1, hENT2, hCNT1, hCNT2, and hCNT3 in both cortex and medulla. Immunoblotting with crude membrane preparations revealed abundant hENT1 and hCNT3 in both cortex and medulla, and little, if any, hENT2, hCNT1, or hCNT2, indicating that the latter were either absent or below limits of detection of immunoassays. hENT1 immunostaining was observed on apical surfaces of proximal tubules and on both apical and basal surfaces of thick ascending loops of Henle and collecting ducts. Prominent hCNT3 immunostaining was observed on apical surfaces of proximal tubules and thick ascending loops of Henle in addition to some cytoplasmic staining. Equilibrium binding of [(3)H]nitrobenzylmercaptopurine ribonucleoside (NBMPR), a high-affinity inhibitor of hENT1, to brush-border membrane vesicles from cortex confirmed the presence of hENT1 on apical surfaces of proximal tubules. Uptake of [(3)H]uridine by polarized renal proximal tubule cells exhibited a sodium-dependent component that was inhibited by thymidine and inosine as well as a sodium-independent component that was partially inhibited by NBMPR and completely inhibited by dilazep, indicating high levels of hENT1 and hCNT3 and low levels of hENT2 activities. The presence of 1) transcripts for hENT1/2 and hCNT1/2/3 and the hENT1 and hCNT3 proteins in human kidneys and 2) hENT1, hENT2, and hCNT3 activities in cultured proximal tubule cells suggest involvement of hENT1, hCNT3, and possibly also hENT2 in renal handling of nucleosides and nucleoside drugs.

Tamoxifen resistance is one of the overarching challenges in the treatment of patients with estrogen receptor (ER)-positive breast cancer. Through a genome-wide RNA interference screen to discover genes responsible for tamoxifen resistance in vitro, we identified insulin-like growth factor binding protein 5 (IGFBP5) as a determinant of drug sensitivity. Specific knockdown of IGFBP5 by retroviral infection with short hairpin RNAexpressing cassette in MCF7 human breast cancer cells (pRS-shIGFBP5) conferred tamoxifen resistance in vitro due to concomitant loss of ERα expression and signaling. IGFBP5 expression was also reduced in MCF7 cells selected for tamoxifen resistance in culture (TAMR). Both tamoxifen-resistant MCF7-TAMR and MCF7-pRS-shIGFBP5 cells could be resensitized to drug by treatment with exogenous recombinant IGFBP5 (rIGFBP5) protein. Treatment with rIGFBP5 protein in mouse tumor xenografts reversed the in vivo tamoxifen resistance of MCF7-pRS-shIGFBP5 cell-derived tumors by reducing tumor cell proliferation. IGFBP5 immunohistochemical staining in a cohort of 153 breast cancer patients showed that low IGFBP5 expression was associated with shorter overall survival after tamoxifen therapy. Thus, IGFBP5 warrants investigation as an agent to reverse tamoxifen resistance.

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The p53 tumor suppressor induces apoptosis in response to genotoxic and environmental stresses. Separately from its functions as a transcription factor, it is also capable to be translocated to the mitochondria and plays a critical role in transcription-independent mitochondrial apoptosis. We previously demonstrated that Tid1 interacts with p53, resulting in mitochondrial translocation of the complex and induction of intrinsic apoptosis [1]; however, the mechanism how they interact has been unknown. In this study, far western analyses demonstrated that Tid1 directly interacted with p53. Using domain deletion mutant constructs, we determined that DnaJ domain of Tid1 was necessary for the interaction, while either Nor C-terminal domains of p53 were sufficient for the interaction. In breast cancer cells, depletion of Tid1 by short hairpin RNA (shRNA) led to absence of p53 accumulation at mitochondria and resistance to apoptosis under hypoxic or genotoxic stresses. Our studies imply that Tid1 could be important in the potential combination chemotherapies of p53-related cancers.