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MDC1 is critical component of the DNA damage response (DDR) machinery and orchestrates the ensuring assembly of the DDR protein at the DNA damage sites, and therefore loss of MDC1 results in genomic instability and tumorigenicity. However, the molecular mechanisms controlling MDC1 expression are currently unknown. Here, we show that miR-22 inhibits MDC1 translation via direct binding to its 3′ untranslated region, leading to impaired DNA damage repair and genomic instability. We demonstrated that activated Akt1 and senescence hinder DDR function of MDC1 by upregulating endogenous miR-22. After overexpression of constitutively active Akt1, homologous recombination was inhibited by miR-22–mediated MDC1 repression. In addition, during replicative senescence and stress-induced premature senescence, MDC1 was downregulated by upregulating miR-22 and thereby accumulating DNA damage. Our results demonstrate a central role of miR-22 in the physiologic regulation of MDC1-dependent DDR and suggest a molecular mechanism for how aberrant Akt1 activation and senescence lead to increased genomic instability, fostering an environment that promotes tumorigenesis. Cancer Res; 75(7); 1298–310. ©2015 AACR.

Supplemental materials and methods and legends for all supplemental figures.

Supplementary Table S1. miRNA predicted to bind the 3`UTR of the MDC1 gene by at least 4 algorithms.

Supplementary Figures S1-S10. Supplementary Figure S1. miR-22 negatively regulates MDC1 expression. Supplementary Figure S2. The effect of miR-22 on the IR-induced nuclear foci of MDC1, 53BP1, and BRCA1 in U2OS cells. Supplementary Figure S3. Reintroduction of MDC1 rescues miR-22-mediated inhibition of DSB repair in U2OS cells. Supplementary Figure S4. Genome-wide DNA copy number variation in the miR-22-overexpressing GM00637 cells. Supplementary Figure S5. miR-22 levels frequently increased in prostate cancer tissues with high pAkt and low MDC1 expression. Supplementary Figure S6. Busufan-induced premature senescence leads to suppression of MDC1 expression. Supplementary Figure S7. Impairment of IR-induced MDC1 foci formation in senescent cells. Supplementary Figure S8. Decline of DNA double-strand break (DSB) repair efficiency in senescent cells. Supplementary Figure S9. Rescue of MDC1 expression in senescent MRC-5 and IMR-90 cells by transfection of miR-22-insensitive MDC1. Supplementary Figure S10. Overexpression of miR-22 induces senescent-like phenotypes in human cancer cells.

Oncogenic Ras mutations are frequently observed in solid tumor cells and are present in pancreatic cancer, colorectal cancer (CRC), and lung cancer (LC) [1, 2]. Mutational activation of K-Ras in these tissues is sufficient to initiate neoplasia in mice [3–5]. Thus, understanding the mechanisms behind Ras-induced oncogenesis is an important goal in cancer therapy. A significant consequence of Ras-mediated signal transduction is the altered expression of a large number of genes. It is well established that canonical Ras signaling is coupled to transcriptional regulation through the activation of the MAPK cascade, which involves the sequential phosphorylation and activation of the serine/threonine kinases RAF, MEK1/2, and ERK1/2 [6–12]. More recently, new regulatory factors that directly interact with K-Ras were identified and shown to play a crucial role in the full range of K-Ras oncogenic phenotypes [13–19]. Despite decades of effort, many aspects of the molecular mechanism underlying Ras-induced tumorigenesis and possible therapeutic targeting remain difficult to identify. Ephexin1 is a member of the DbI family of guanine nucleotide exchange factors (GEFs) and serves as a direct link between Eph receptors and the Rho-family of GTPases [20, 21]. Ephexin1 is highly expressed in the nervous system during development and is involved in many neurophysiological events [22–26]. It is phosphorylated by the Src family of kinases and by fibroblast growth factor receptor signaling, leading to the regulation of GEF activity, which in turn regulates actin cytoskeletal dynamics [22, 27]. Through extensive analysis of the gene expression patterns in cells with activated forms of Ras and Rho families, it was found that Ephexin1 was highly upregulated by H-RasG12V in NIH3T3 cells [28]. In humans, Ephexin1 is upregulated in papillary thyroid cancers (PTC) and has been identified as the most reliable marker for PTC diagnosis [29]. Therefore, the implication of these results is that Ephexin1 may be involved in tumor proliferation and progression, particularly in cancer cells with oncogenic Ras mutations. However, the functional consequences of Ephexin1 had not yet been addressed. In the present study, we show that Ephexin1 is overexpressed in both CRC and LC tissues and is associated with a poor prognosis for both cancers, and provide evidence for the functional and clinical significance of Ephexin1 and identify a potential therapeutic target for the case of CRC and LC caused by Ras mutations.

Human CtIP maintains genomic integrity primarily by promoting 5′ DNA end resection, an initial step of the homologous recombination (HR). A few mechanisms have been suggested as to how CtIP recruitment to damage sites is controlled, but it is likely that we do not yet have full understanding of the process. Here, we provide evidence that CtIP recruitment and functioning are controlled by the SIAH2 E3 ubiquitin ligase. We found that SIAH2 interacts and ubiquitinates CtIP at its N-terminal lysine residues. Mutating the key CtIP lysine residues impaired CtIP recruitment to DSBs and stalled replication forks, DSB end resection, overall HR repair capacity of cells, and recovery of stalled replication forks, suggesting that the SIAH2-induced ubiquitination is important for relocating CtIP to sites of damage. Depleting SIAH2 consistently phenocopied these results. Overall, our work suggests that SIAH2 is a new regulator of CtIP and HR repair, and emphasizes that SIAH2-mediated recruitment of the CtIP is an important step for CtIP’s function during HR repair.

The cell signaling factors EGFR, EphA2, and Ephexin1 are associated with lung and colorectal cancer and play an important role in tumorigenesis. Although the respective functional roles of EGFR and EphA2 are well known, interactions between these proteins and a functional role for the complex is not understood. Here, we showed that Ephexin1, EphA2, and EGFR are each expressed at higher levels in lung and colorectal cancer patient tissues, and binding of EGFR to EphA2 was associated with both increased tumor grade and metastatic cases in both cancer types. Treatment with Epidermal Growth Factor (EGF) induced binding of the RR domain of EGFR to the kinase domain of EphA2, and this binding was promoted by Ephexin1. Additionally, the AKT-mediated phosphorylation of EphA2 (at Ser897) promoted interactions with EGFR, pointing to the importance of this pathway. Two mutations in EGFR, L858R and T790M, that are frequently observed in lung cancer patients, promoted binding to EphA2, and this binding was dependent on Ephexin1. Our results indicate that the formation of a complex between EGFR, EphA2, and Ephexin1 plays an important role in lung and colorectal cancers, and that inhibition of this complex may be an effective target for cancer therapy.

Objective: To investigate a feasible candidate for an appropriate cell line for the orthotopic renal cell carcinoma (RCC) model. Methods: Normal human proximal tubule cells (HK-2) and RCC cells were used for MTT assay, Western blotting, sphere-forming assay, and orthotopic injection of BALB/c-nude mice. Immunohistochemistry was adopted in tissue arrays and orthotopic tumors. Results: Primary RCC cells showed resistance to a GPX4 inhibitor compared to HK-2 and to metastatic RCC cells, Caki-1. Caki-2 and SNU-333 cells showed resistance to ferroptosis via increased GPX4 and FTH1, respectively. RCC cells showed increased αSMA, in which Caki-2 and SNU-333 cells exhibited different epithelial–mesenchymal transition and cancer stem cell markers. Caki-1 and SNU-333 cells formed spheres in vitro and orthotopic tumor masses in vivo. The injected SNU-333 tumor only showed high intensities of CD10 and PAX8, markers of renal origin. Conclusion: SNU-333 cell line exhibited resistance via iron metabolism and stemness, and had tumor-initiating capacities in vitro and in vivo. These results suggest that among the cells tested, SNU-333 cells were the most promising for the establishment of an orthotopic RCC model for further researches.

The Hsp70-binding protein 1 (HspBP1) belongs to a family of co-chaperones that regulate Hsp70 activity and whose biological significance is not well understood. In the present study, we show that when HspBP1 is either knocked down or overexpressed in BRCA1-proficient breast cancer cells, there were profound changes in tumorigenesis, including anchorage-independent cell growth in vitro and in tumor formation in xenograft models. However, HspBP1 did not affect tumorigenic properties in BRCA1-deficient breast cancer cells. The mechanisms underlying HspBP1-induced tumor suppression were found to include interactions with BRCA1 and promotion of BRCA1-mediated homologous recombination DNA repair, suggesting that HspBP1 contributes to the suppression of breast cancer by regulating BRCA1 function and thereby maintaining genomic stability. Interestingly, independent of BRCA1 status, HspBP1 facilitates cell survival in response to ionizing radiation (IR) by interfering with the association of Hsp70 and apoptotic protease-activating factor-1. These findings suggest that decreased HspBP1 expression, a common occurrence in high-grade and metastatic breast cancers, leads to genomic instability and enables resistance to IR treatment.

From the evolutionary myology, the additional tendon of the extensor hallucis longus (EHL) muscle represents the sample of a new acquisition. We aimed to determine whether the insertion pattern of the EHL muscle differs in Koreans according to demographic populations, especially between Jeju islanders and the Korean Peninsula inhabitants. We used 69 Korean cadavers and classified the tendinous insertion of the EHL muscle as Pattern I, Pattern II, and Pattern III. The ratio of each Pattern in adult cadaveric samples was compared between demographic populations. The proportion of Pattern I, Pattern II, and Pattern III of the EHL muscle was 30.43, 63.77, and 5.80%, respectively, further divided into 18.00 vs. 36.04%, 72.00 vs. 60.47%, 10.00 vs. 3.49% in Jeju islanders vs. peninsular Koreans. There was a considerable difference in the insertion patterns of the EHL muscle in each regional group (p = 0.032), but not in each gender, age, and body sides of lower limbs. The findings of this study indicate that there was a higher incidence of the accessory tendon(s) of the EHL muscle in Koreans and the distributed insertion patterns of the EHL muscle was significantly different between Jeju islanders and peninsular Koreans.

Background Although renal cell carcinoma (RCC) is known to be susceptible to ferroptosis, we found primary RCC cells showed resistance to ferroptosis and aimed to investigate a feasible candidate for an appropriate cell line for the RCC model. Methods Glutathione peroxidase 4 (GPX4) immunostaining was adopted in the RCC tissue microarrays. Normal human proximal tubule cells (HK-2) and RCC cell lines were used for the MTT assay, Western blotting, sphere-forming assay, and orthotopic injection of athymic Balb/c-nude mice. Results GPX4 immunostaining showed low intensity compared to the normal kidney, which coincided with the ferroptosis-susceptibility of RCC. Primary RCC cell lines (Caki-2, SNU-333, SNU-349, and SNU-1272) showed resistance to 5-fluorouracil and a GPX4 inhibitor compared to the HK-2 cells and to metastatic RCC cells (Caki-1). The Caki-2 cells showed increased GPX4 and xCT, and the SNU-333 cells showed increased ferritin heavy chain (FTH1) compared to the other RCC cells. The Caki-2 cells showed increased aSMA, fibronectin, vimentin, and SNAIL, and the SNU-333 cells showed increased aSMA, E-cadherin, and EpCAM. The Caki-2 cells showed increased Sox-2 and CD105, and the SNU-333 cells showed increased c-Myc and Lgr5. The Caki-1 and SNU-333 cells formed spheres in vitro and orthotopic RCC masses in vivo. The injected SNU-333 tumor only showed high intensities of CD10 and PAX8, consistent with the diagnostic criteria for RCC. Conclusions The primary RCC cell lines used in this study were more resistant to ferroptosis and 5-fluorouracil, and the SNU-333 cells showed tumor-initiating capacities in vitro and in vivo. These results suggest that SNU-333 might be suitable as a orthotopic RCC model for future research.

Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin &alpha, 2 (KPNA2), a nucleocytoplasmic transport adaptor, and showed that KPNA2 is necessary for MDC1 nuclear import. Thereafter, we identified a functional nuclear localization signal (NLS) between amino acid residues 1989&ndash, 1994 of the two Breast Cancer 1 (BRCA1) carboxyl-terminal (tBRCT) domain of MDC1 and demonstrated disruption of this NLS impaired interaction between MDC1 and KPNA2 and reduced nuclear localization of MDC1. In KPNA2-depleted cells, the recruitment of MDC1, along with the downstream signaling p roteins Ring Finger Protein 8 (RNF8), 53BP1-binding protein 1 (53BP1), BRCA1, and Ring Finger Protein 168 (RNF168), to DNA damage sites was abolished. Additionally, KPNA2-depleted cells had a decreased rate of homologous recombination (HR) repair. Our data suggest that KPNA2-mediated MDC1 nuclear import is important for DDR signaling and DSB repair.

In the present study, we investigated the effect of oncogenic H-Ras on rat mdr1b expression in NIH3T3 cells. The constitutive expression of H-RasV12 was found to downregulate the mdr1b promoter activity and mdr1b mRNA expression. The doxorubicin-induced mdr1b promoter activity of the H-RasV12 expressing NIH3T3 cells was markedly lower than that of control NIH3T3 cells. Additionally, there is a positive correlation between the level of H-RasV12 expression and a sensitivity to doxorubicin toxicity. To examine the detailed mechanism of H-RasV12-mediated down-regulation of mdr1b expression, antioxidant N-acetylcysteine (NAC) and NADPH oxidase inhibitor diphenylene iodonium (DPI) were used. Pretreating cells with either NAC or DPI significantly enhanced the oncogenic H-Ras-mediated down-regulation of mdr1b expression and markedly prevented doxorubicin-induced cell death. Moreover, NAC and DPI treatment led to a decrease in ERK activity, and the ERK inhibitors PD98059 or U0126 enhanced the mdr1b-Luc activity of H-RasV12-NIH3T3 and reduced doxorubicin-induced apoptosis. These data suggest that RasV12 expression could downregulate mdr1b expression through intracellular reactive oxygen species (ROS) production, and ERK activation induced by ROS, is at least in part, contributed to the downregulation of mdr1b expression.

MDC1 plays a critical role in the DNA damage response (DDR) by interacting directly with several factors including γ-H2AX. However, the mechanism by which MDC1 is recruited to damaged sites remains elusive. Here, we show that MDC1 interacts with a helix–loop–helix (HLH)-containing protein called inhibitor of DNA-binding 3 (ID3). In response to double-strand breaks (DSBs) in the genome, ATM phosphorylates ID3 at serine 65 within the HLH motif, and this modification allows a direct interaction with MDC1. Moreover, depletion of ID3 results in impaired formation of ionizing radiation (IR)-induced MDC1 foci, suppression of γ-H2AX-bound MDC1, impaired DSB repair, cellular hypersensitivity to IR, and genomic instability. Disruption of the MDC1–ID3 interaction prevents accumulation of MDC1 at sites of DSBs and suppresses DSB repair. Thus, our study uncovers an ID3-dependent mechanism of recruitment of MDC1 to DNA damage sites and suggests that the ID3–MDC1 interaction is crucial for DDR. MDC1 is a key component of the DNA damage response and interacts with several factors such as γ-H2AX. Here the authors show that MDC1 interacts with ID3, facilitating MDC1 recruitment to sites of damage and repair of breaks.

MDC1 plays a critical role in the DNA damage response (DDR) by interacting directly with several factors including γ-H2AX. However, the mechanism by which MDC1 is recruited to damaged sites remains elusive. Here, we show that MDC1 interacts with a helix–loop–helix (HLH)-containing protein called inhibitor of DNA-binding 3 (ID3). In response to double-strand breaks (DSBs) in the genome, ATM phosphorylates ID3 at serine 65 within the HLH motif, and this modification allows a direct interaction with MDC1. Moreover, depletion of ID3 results in impaired formation of ionizing radiation (IR)-induced MDC1 foci, suppression of γ-H2AX-bound MDC1, impaired DSB repair, cellular hypersensitivity to IR, and genomic instability. Disruption of the MDC1–ID3 interaction prevents accumulation of MDC1 at sites of DSBs and suppresses DSB repair. Thus, our study uncovers an ID3-dependent mechanism of recruitment of MDC1 to DNA damage sites and suggests that the ID3–MDC1 interaction is crucial for DDR., MDC1 is a key component of the DNA damage response and interacts with several factors such as γ-H2AX. Here the authors show that MDC1 interacts with ID3, facilitating MDC1 recruitment to sites of damage and repair of breaks.

Objectives To analyze the urothelial responses to mitomycin C treatment after urethral injury in rats, as the urothelium might play a role in the pathogenesis of urethral stricture. Methods Male Sprague–Dawley rats were divided into four groups (n = 5/group): negative control, positive control without further treatment, experimental control treated with sodium hyaluronate and sodium carboxymethylcellulose, and experimental treated with mitomycin C after internal urethrotomy. Results Compared with negative controls, positive controls showed a significant increase in cell proliferation and DNA damage accompanied by a considerable decrease in DNA repair in the urothelium, which resulted in urethral stricture. Experimental controls showed a significant increase in cell proliferation, DNA damage and DNA repair compared with negative controls. The mitomycin C-treated group showed a significant decrease in cell proliferation and DNA damage, but a considerable increase in DNA repair compared with the positive and experimental control groups. DNA damage was immediately increased after urethral injury, but DNA repair and cell proliferation showed belated and upregulated expression after mitomycin C treatment. Conclusions Mitomycin C could induce healthy re-epithelialization without severe damage in the urothelium. This finding might support the possibility of using mitomycin C as an adjuvant therapy for urethral strictures, and it might also suggest a urothelial role in the process of urethral stricture after urethral injury.

MDC1 is critical component of the DNA damage response (DDR) machinery and orchestrates the ensuring assembly of the DDR protein at the DNA damage sites, and therefore loss of MDC1 results in genomic instability and tumorigenicity. However, the molecular mechanisms controlling MDC1 expression are currently unknown. Here, we show that miR-22 inhibits MDC1 translation via direct binding to its 3′ untranslated region, leading to impaired DNA damage repair and genomic instability. We demonstrated that activated Akt1 and senescence hinder DDR function of MDC1 by upregulating endogenous miR-22. After overexpression of constitutively active Akt1, homologous recombination was inhibited by miR-22–mediated MDC1 repression. In addition, during replicative senescence and stress-induced premature senescence, MDC1 was downregulated by upregulating miR-22 and thereby accumulating DNA damage. Our results demonstrate a central role of miR-22 in the physiologic regulation of MDC1-dependent DDR and suggest a molecular mechanism for how aberrant Akt1 activation and senescence lead to increased genomic instability, fostering an environment that promotes tumorigenesis. Cancer Res; 75(7); 1298–310. ©2015 AACR.

The START domain-containing 6 (StarD6) was originally reported to play a role during male germ cell maturation. We have since reported on StarD6 in the developing hypothyroid rat brain. Therefore, we investigated qualitative and quantitative changes of StarD6 in the aging rat brain and testes of male Sprague-Dawley rats. Serum testosterone levels decreased with aging and total protein levels of StarD6 in the testes decreased. While the immunolocalization of StarD6 in the spermatocytes decreased, cytoplasmic localization appeared in the aged testes. Compared with young rats, aged rats showed decreased StarD6 in the cerebrum and cerebellum without changes in immunolocalization in the cortical neurons of the cerebral cortex and Purkinje cells of the cerebellar cortex. Aged rats also showed increases in StarD6 in the hippocampus with changes in its immunolocalization from the Stratum pyramidale to the Stratum radiatum and Stratum lacunosum-moleculare. Taken together, StarD6 decreased with aging in the testes, which implies that StarD6 might play a role in impaired spermatogenesis in the aged rat. StarD6 decreased in the cerebrum and the cerebellum, but slightly increased in the hippocampus, which suggests that StarD6 might also play a role for neurosteroidogenesis in the hippocampus of aged rats.

Ephexin-1 functions as guanine nucleotide exchange factors for the Rho-type GTPases which have important roles in neuronal development including axon guidance, migration, morphogenesis, and plasticity of neurons. As little is known about ephexin-1 in the cerebellum, we investigated the immunolocalization of ephexin-1 in the developing canine cerebellum. While the cellular maturation was followed by the temporal pattern, the calbindin D-28k and ephexin-1 immunoreactivities gradually increased in developing canine cerebellum. When compared to the calbindin D-28k immunoreactivities, belated ephexin-1 immunolocalization was observed in the Purkinje cells which aligned a single layer during cerebellar development. These results suggest that ephexin-1 might play an important role in the development of the Purkinje cells during the first two postnatal weeks based on its immunolocalization in the present study.

The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65–171 is critical for p53R2–MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity.

Interstitial cells of Cajal (ICCs) are the pacemaking cells in the gastrointestinal muscles that generate the rhythmic oscillations in membrane potential known as slow waves. ICCs also mediate or transduce inputs from the enteric nervous system. Substance P (SubP) is a member of the family of mammalian tachykinin peptides that are predominantly released by enteric neurons. This study assessed the relationship of Na(+)-leak channel (NALCN) in the SubP-induced depolarization in pacemaking activity in the gastrointestinal tract. The patch-clamp technique for whole-cell recording was used in cultured cluster and single ICCs. Electrophysiological and pharmacological properties of SubP in ICC pacemaking activity were similar to those of NALCN. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of NALCN messenger RNA and protein in mouse small intestine. NALCN is involved in the SubP-induced depolarization of intestinal pacemaking activity. The protein is a potential target for pharmacological treatment of motor disorders of the gut.

Unlike steroidogenic acute regulatory protein (StAR), one of the cholesterol transport protein, little attention is given to StarD6 which belongs to a family of StAR-related lipid transfer domain proteins. Although we undertook previous works with StarD6 in the nervous system, the characteristics are in controversy to date. Therefore, we attempted to investigate the morphological characteristics of StarD6 in the nervous system are the same as StAR in vitro and in vivo. The number of immunoreactive cells was significantly different by StAR or StarD6 in the cultured glioblastoma cell lines and dopaminergic neuronal cell lines. StarD6 immunoreactivity was changed by the presence of DNA-dependent protein kinase, while the dependency was not observed in StAR immunoreactivity. Besides, StarD6 was mainly observed in the stratum pyramidale and StAR in the other strata of normal rat hippocampus proper. Increased immunolocalization of StAR and StarD6 was seen in the stratum pyramidale and the strata lacunosum-moleculare, respectively, 3 h after pilocarpine-induced epilepsy. Taken together, morphological aspects of StarD6 were significantly different from those of StAR in cultured glial and neuronal cells, as well as the distribution in the normal and epileptic rat hippocampus. These results suggested that StarD6 did not mark the same as StAR in vitro and in vivo.

MSH6, a key component of the MSH2-MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70.

Although the accumulation of 8-oxo-dGTP in DNA is associated with apoptotic cell death and mutagenesis, little is known about the exact mechanism of hMTH1-mediated suppression of oxidative-stress-induced cell death. Therefore, we investigated the regulation of DNA-damage-related apoptosis induced by oxidative stress using control and hMTH1 knockdown cells. Small interfering RNA (siRNA) was used to suppress hMTH1 expression in p53-proficient GM00637 and H460 cells, resulting in a significant increase in apoptotic cell death after H(2)O(2) exposure; however, p53-null, hMTH1-deficient H1299 cells did not exhibit H(2)O(2)-induced apoptosis. In addition, hMTH1-deficient GM00637 and H460 cells showed increased caspase-3/7 activity, cleaved caspase-8, and Noxa expression, and gamma-H2AX formation in response to H(2)O(2). In contrast, the caspase inhibitors, p53-siRNA, and Noxa-siRNA suppressed H(2)O(2)-induced cell death. Moreover, in 8-week (long-term) cultured H460 and H1299 cells, hMTH1 suppression increased cell death, Noxa expression, and gamma-H2AX after H(2)O(2) exposure, compared to 3-week (short-term) cultured cells. These data indicate that hMTH1 plays an important role in protecting cells against H(2)O(2)-induced apoptosis via a Noxa- and caspase-3/7-mediated signaling pathway, thus conferring a survival advantage through the inhibition of oxidative-stress-induced DNA damage.

DNA damage and mutations in the genome increase with age. To determine the potential mechanisms of senescence-dependent increases in genomic instability, we analyzed DNA mismatch repair (MMR) efficiency in young and senescent human colonic fibroblast and human embryonic lung fibroblast. It was found that MMR activity is significantly reduced in senescent cells. Western blot and immunohistochemistry analysis revealed that hMSH2 and MSH6 protein (MutSα complex), which is a known key component in the MMR pathway, is markedly down-regulated in senescent cells. Moreover, the addition of purified MutSα to extracts from senescent cells led to the restoration of MMR activity. Semiquantitative reverse transcription-PCR analysis exhibited that MSH2 mRNA level is reduced in senescent cells. In addition, a decrease in E2F transcriptional activity in senescent cells was found to be crucial for MSH2 suppression. E2F1 small interfering RNA expression reduced hMSH2 expression and MMR activity in young human primary fibroblast cells. Importantly, expression of E2F1 in quiescent cells restored the MSH2 expression as well as MMR activity, whereas E2F1-infected senescent cells exhibited no restoration of MSH2 expression and MMR activity. These results indicate that the suppression of E2F1 transcriptional activity in senescent cells lead to stable repression of MSH2, followed by a induction of MutSα dysfunction, which results in a reduced cellular MMR capacity in senescent cells. (Mol Cancer Res 2008;6(6):978–89)

We demonstrate that KIOM-79, combined extracts obtained from Magnolia officinalis, Pueraria lobata, Glycyrrhiza uralensis, and Euphorbia pekinensis, inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells. Treatment of RAW 264.7 cells with KIOM-79 inhibited LPS-stimulated nitric oxide production in a dose-related manner. Immunohisto-chemical staining of iNOS and RT-PCR analysis showed that the decrease of NO was due to the inhibition of iNOS gene expression. Immunostaining of p65, EMSA, and reporter gene assay showed that KIOM-79 inhibited NF-κ/Rel nuclear translocation, DNA binding, and transcriptional activation, respectively. Western immunoblot analysis of p38 kinase showed KIOM-79 significantly inhibited the phosphoylation of p38 kinase which is important in the regulation of iNOS gene expression. Collectively, this series of experiments indicates that KIOM inhibits iNOS gene expression by blocking NF-κ/Rel and p38 kinase signaling. Due to the critical role that NO release plays in mediating inflammatory responses, the inhibitory effects of KIOM-79 on iNOS suggest that KIOM-79 may represent a useful anti-inflammatory agent.

1 We studied the modulation of pacemaker activities by bradykinin in cultured interstitial cells of Cajal (ICC) from murine small intestine with the whole-cell patch-clamp technique. Externally applied bradykinin produced membrane depolarization in the current-clamp mode and increased tonic inward pacemaker currents in the voltage-clamp mode. 2 Pretreatment with bradykinin B1 antagonist did not block the bradykinin-induced effects on pacemaker currents. However, pretreatment with bradykinin B2 antagonist selectively blocked the bradykinin-induced effects. Also, only externally applied selective bradykinin B2 receptor agonist produced tonic inward pacemaker currents and ICC revealed a colocalization of the bradykinin B2 receptor and c-kit immunoreactivities, but bradykinin B1 receptors did not localize in ICC. 3 External Na+-free solution abolished the generation of pacemaker currents and inhibited the bradykinin-induced tonic inward current. However, a Cl− channel blocker (DIDS) did not block the bradykinin-induced tonic inward current. 4 The pretreatment with Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the bradykinin-induced action. 5 Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the bradykinin-induced effects on pacemaker currents. 6 These results suggest that bradykinin modulates the pacemaker activities through bradykinin B2 receptor activation in ICC by external Ca2+ influx and internal Ca2+ release via protein kinase C- or cyclooxygenase-independent mechanism. Therefore, the ICC are targets for bradykinin and their interaction can affect intestinal motility. British Journal of Pharmacology (2006) 148, 918–926. doi:10.1038/sj.bjp.0706806

We demonstrate that polysaccharides isolated from Salicornia herbacea (Salicornia polysaccharides, SPS) significantly induces nitric oxide (NO) production and inducible NO synthase (iNOS) transcription through the activation of nuclear factor-kappaB/Rel (NF-kappaB/Rel). SPS dose-dependently induced the production of NO in isolated mouse peritoneal macrophages and RAW 264.7, a mouse macrophage-like cell line. Moreover, iNOS gene expression was strongly induced by SPS in RAW 264.7 cells. To further investigate the mechanism responsible for the induction of iNOS gene expression, we investigated the effect of SPS on the activation of transcription factors including NF-kappaB/Rel and Oct, whose binding sites were located in the promoter of iNOS gene. Treatment of RAW 264.7 cells with SPS produced strong induction of NF-kappaB/Rel-dependent reporter gene expression, whereas Oct-dependent gene expression was not affected by SPS. Nuclear translocation and DNA binding activity of NF-kappaB/Rel was significantly induced by SPS. The treatment with NF-kappaB SN50, an inhibitor of NF-kappaB/Rel nuclear translocation, effectively inhibited the activation of NF-kappaB/Rel binding complexes and NO production. In conclusion, we demonstrate that SPS stimulates macrophages to express iNOS gene through the activation of NF-kappaB/Rel.

The interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract and generate an electrical rhythm in gastrointestinal muscles. We investigated the possibility that PGE2 might affect the electrical properties of cultured ICC by activating ATPdependent K+ channels and, the EP receptor subtypes and the subunits of ATP-dependent K+ channels involved in these activities were identified. In addition, the regulation of intracellular Ca2+ ([Ca2+]i) mobilization may be involved the action of PGE2 on ICC. Treatments of ICC with PGE2 inhibited electrical pacemaker activities in the same manner as pinacidil, an ATPdependent K+ channel opener and PGE2 had only a dose-dependent effect. Using RT-PCR technique, we found that ATP-dependent K+ channels exist in ICC and that these are composed of Kir 6.2 and SUR 2B subunits. To characterize the specific membrane EP receptor subtypes in ICC, EP receptor agonists and RT-PCR were used: Butaprost (an EP2 receptor agonist) showed the actions on pacemaker currents in the same manner as PGE2. However sulprostone (a mixed EP1 and EP3 agonist) had no effects. In addition, RT-PCR results indicated the presence of the EP2 receptor in ICC. To investigate cAMP involvement in the effects of PGE2 on ICCs, SQ-22536 (an inhibitor of adenylate cyclase) and cAMP assays were used. SQ-22536 did not affect the effect of PGE2 on pacemaker currents, and PGE2 did not stimulate cAMP production. Also, we found PGE2 inhibited the spontaneous [Ca2+]i oscillations in cultured ICC. These observations indicate that PGE2 alters pacemaker currents by activating the ATP-dependent K+ channels comprised of Kir 6.2-SUR 2B in ICC and this action of PGE2 are through EP2 receptor subtype and also the activation of ATP-dependent K+ channels involves intracellular Ca2+ mobilization.

To analyze the urothelial responses to mitomycin C treatment after urethral injury in rats, as the urothelium might play a role in the pathogenesis of urethral stricture.Male Sprague-Dawley rats were divided into four groups (n = 5/group): negative control, positive control without further treatment, experimental control treated with sodium hyaluronate and sodium carboxymethylcellulose, and experimental treated with mitomycin C after internal urethrotomy.Compared with negative controls, positive controls showed a significant increase in cell proliferation and DNA damage accompanied by a considerable decrease in DNA repair in the urothelium, which resulted in urethral stricture. Experimental controls showed a significant increase in cell proliferation, DNA damage and DNA repair compared with negative controls. The mitomycin C-treated group showed a significant decrease in cell proliferation and DNA damage, but a considerable increase in DNA repair compared with the positive and experimental control groups. DNA damage was immediately increased after urethral injury, but DNA repair and cell proliferation showed belated and upregulated expression after mitomycin C treatment.Mitomycin C could induce healthy re-epithelialization without severe damage in the urothelium. This finding might support the possibility of using mitomycin C as an adjuvant therapy for urethral strictures, and it might also suggest a urothelial role in the process of urethral stricture after urethral injury.

Background & Aims: Interstitial cells of Cajal are pacemakers in the gastrointestinal tract, regulating rhythmicity by activating nonselective cation channels. In Caenorhabditis elegans, the melastatin-type transient receptor potential (TRPM) channel, especially TRPM7, was suggested as being involved in defecation rhythm. The aim here was to show that the nonselective cation channel in interstitial cells of Cajal in mouse small intestine has properties essentially identical to those of murine TRPM7, heterologously expressed in human embryonic kidney cells. Methods: The patch-clamp technique for whole-cell recording was used in cultured or single interstitial cells of Cajal. TRPM7-specific small interfering RNAs were used for specific inhibition of TRPM7. Results: Electrophysiological and pharmacological properties of the nonselective cation channel in interstitial cells of Cajal were the same as those of TRPM7. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of TRPM7 messenger RNA and protein in mouse small intestine. Treatment of primary cultured interstitial cells of Cajal with TRPM7-specific small interfering RNA resulted in inhibition of pacemaking activity. Conclusions: TRPM7 is required for intestinal pacemaking. The protein is a likely potential target for pharmacological treatment of motor disorders of the gut.

The multifunctional mammalian apurinic/apyrimidinic (AP) endonuclease (APE) participates in the repair of AP sites in the cellular DNA as well as participating in the redox regulation of the transcription factor function. The function of APE is considered as the rate-limiting step in DNA base excision repair. Paradoxically, an unbalanced increase in APE protein leads to genetic instability. Therefore, we investigated the mechanisms of genetic instability that are induced by APE. Here, we report that the overexpression of APE protein disrupts the repair of DNA mismatches, which results in microsatellite instability (MSI). We found that expression of APE protein led to the suppression of the repair of DNA mismatches in the normal human fibroblast cells. Western blot analysis revealed that hMSH6 protein was markedly reduced in the APE-expressing cells. Moreover, the addition of purified Mutalpha (MSH2 and MSH6 complex) to the extracts from the APE-expressing cells led to the restoration of mismatch repair (MMR) activity. By performing MMR activity assay and MSI analysis, we found that the co-expression of hMSH6 and APE exhibited the microsatellite stability, whereas the expression of APE alone generated the MSI-high phenotype. The APE-mediated decrease in MMR activity described here demonstrates the presence of a new and highly effective APE-mediated mechanism for MSI.

Cadmium is a well known human and animal carcinogen and is a ubiquitous contaminant in the environment. Although the carcinogenic mechanism of cadmium is a multifactorial process, oxidative DNA damage is believed to be of prime importance. In particular, cadmium suppresses the capacity of cells to repair oxidative DNA damage. In this study, cadmium treatment led to a significant increase in gamma-ray-induced 8-oxoguanine (8-oxoG) formation. Western blotting and semiquantitative reverse transcription-PCR revealed that cadmium treatment caused a decrease in the expression level of human OGG1 (8-oxoguanine-DNA glycosylase-1; hOGG1) in human fibroblast GM00637 and HeLa S3 cells. In addition, the cadmium-mediated decrease in hOGG1 transcription was the result of decreased binding of the transcription factor Sp1 to the hOGG1 promoter. Finally, we show that an increase in the functional hOGG1 expression level could inhibit the cadmium-mediated increase in gamma-ray-induced 8-oxoG accumulation as well as in gamma-radiation-induced mutation frequency at the HPRT (hypoxanthine-guanine phosphoribosyltransferase) gene locus. These results suggest that cadmium attenuates removal of gamma-ray-induced 8-oxoG adducts, which in turn increases the mutation frequency, and that this effect might, at least in part, result from suppression of hOGG1 transcription via inactivation of Sp1 as a result of cadmium treatment.

Bcl-2 stimulates mutagenesis after the exposure of cells to DNA-damaging agents. However, the biological mechanisms of Bcl-2-mediated mutagenesis have remained largely obscure. Here we demonstrate that the Bcl-2-mediated suppression of hMSH2 expression results in a reduced cellular capacity to repair mismatches. The pathway linking Bcl-2 expression to the suppression of mismatch repair (MMR) activity involves the hypophosphorylation of pRb, and then the enhancement of the E2F-pRb complex. This is followed by a decrease in hMSH2 expression. MMR has a key role in protection against deleterious mutation accumulation and in maintaining genomic stability. Therefore, the decreased MMR activity by Bcl-2 may be an underlying mechanism for Bcl-2-promoted oncogenesis.

Metallothioneins (MT) play an important biological role in preventing oxidative damage to cells. We have previously demonstrated that the efficiency of the protective effect of MT-III against the DNA degradation from oxidative damage was much higher than that of MT-I/II. As an extension of the latter investigation, this study aimed to assess the ability of MT-III to suppress 8-oxoguanine (8-oxoG), which is one of the major base lesions formed after an oxidative attack to DNA and the mutant frequency of the HPRT gene in human fibroblast GM00637 cells upon exposure to gamma-rays. We found that human MT-III expression decreased the level of 8-oxoG and mutation frequency in the gamma-irradiated cells. Using an 8-oxoguanine DNA glycosylase (OGG1)-specific siRNAs, we also found that MT-III expression resulted in the suppression of the gamma-radiation-induced 8-oxoG accumulation and mutation in the OGG1-depleted cells. Moreover, the down-regulation of MT in human neuroblastoma SKNSH cells induced by MT-specific siRNA led to a significant increase in the 8-oxoG level, after exposure to gamma-irradiation. These results suggest that under the conditions of gamma-ray oxidative stress, MT-III prevents the gamma-radiation-induced 8-oxoG accumulation and mutation in normal and hOGG1-depleted cells, and this suppression might, at least in part, contribute to the anticarcinogenic and neuroprotective role of MT-III.

1. Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous inward currents (pacemaker currents) responsible for the production of slow waves in gastrointestinal smooth muscle. The effects of noradrenaline on the pacemaker currents in cultured ICCs from murine small intestine were investigated by using whole-cell patch-clamp techniques at 30 degrees C. 2. Under current clamping, ICCs had a mean resting membrane potential of -58+/-5 mV and produced electrical slow waves. Under voltage clamping, ICCs produced pacemaker currents with a mean amplitude of -410+/-57 pA and a mean frequency of 16+/-2 cycles min(-1). 3. Under voltage clamping, noradrenaline inhibited the amplitude and frequency of pacemaker currents and increased resting currents in the outward direction in a dose-dependent manner. These effects were reduced by intracellular GDP beta S. 4. Noradrenaline-induced effects were blocked by propranolol (beta-adrenoceptor antagonist). However, neither prazosin (alpha(1)-adrenoceptor antagonist) nor yohimbine (alpha(2)-adrenoceptor antagonist) blocked the noradrenaline-induced effects. Phenylephrine (alpha(1)-adrenoceptor agonist) had no effect on the pacemaker currents, whereas isoprenaline (beta-adrenoceptor agonist) mimicked the effect of noradrenaline. Atenolol (beta(1)-adrenoceptor antagonist) blocked the noradrenaline-induced effects, but butoxamine (beta(2)-adrenoceptor antagonist) did not. In addition, BRL37344 (beta(3)-adrenoceptor agonist) had no effect on pacemaker currents. 5. 9-(Tetrahydro-2-furanyl)-9H-purine-6-amine (SQ-22536; adenylate cyclase inhibitor) and a myristoylated protein kinase A inhibitor did not inhibit the noradrenaline-induced effects and 8-bromo-cAMP had no effects on pacemaker currents. 8-Bromo-cGMP and SNAP inhibited pacemaker currents and these effects of SNAP were blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a guanylate cyclase inhibitor). However, ODQ did not block the noradrenaline-induced effects. 6. Neither tetraethylammonium (a voltage-dependent K(+) channel blocker), apamin (a Ca(2+)-dependent K(+) channel blocker) nor glibenclamide (an ATP-sensitive K(+) channel blocker) blocked the noradrenaline-induced effects. 7. The results suggest that noradrenaline-induced stimulation of beta(1)-adrenoceptors in the ICCs inhibits pacemaker currents, and that this is mediated by the activation of G-protein. Neither adenylate cyclase, guanylate cyclase nor a K(+) channel-dependent pathway are involved in this effect of noradrenaline.

The calcium-binding proteins, parvalbumin and calbindin D-28k, are markers of different classes of GABAergic interneurons and display different functions. The present study was attempted to determine immunoreactivities and colocalization of the parvalbumin and calbindin D-28k in the developing canine hippocampus by immunohistochemistry. The calcium-binding protein-containing neurons showed different developmental patterns. The first appearance of parvalbumin immunoreactive nonpyramidal cells was observed at P7. Parvalbumin immunoreactivity was elicited by the sequence from CA3 to CA1 to reach an adult-like distribution pattern, which was reached at P60, while calbindin D-28k immunoreactivity appeared from P0, including pyramidal and nonpyramidal cells. The characteristic distribution of calbindin D-28k immunoreactive pyramidal cells was clarified by P28, and an adult-like distribution pattern was reached by the end of the second postnatal month. Double-labeled nonpyramidal cells were frequently seen in the subareas, CA3 of P14/CA1–CA2 of P28, where parvalbumin immunoreactive nonpyramidal cells were emerging. These data suggest that the colocalization of the two calcium-binding proteins during development is related closely to the area-specific maturation of parvalbumin expression, although either prenatal expression of calbindin D-28k or parvalbumin was not determined.

Mutations in mismatch repair (MMR) genes are commonly associated with the development of colorectal cancer. Additionally, base excision repair, which involves apurinic/apyrimidinic endonuclease 1 (APE1), recognizes and eliminates oxidative DNA damage. Here, we investigated the possible roles of APE1 in dextran sulfate sodium (DSS)-induced acute colitis using the young rat model. Four-week-old Sprague–Dawley rats were administered 2% DSS in drinking water for 1 week. MMR and APE1 expression levels were assessed by western blotting and immunohistochemistry. Following DSS treatment, growth of young rats failed and the animals had loose stools. Together with the histological changes associated with acute colitis, APE1 and MSH2 levels increased significantly at 3 and 5 days after DSS treatment, respectively. The difference between APE1 and MSH2 expression was significant. DSS-induced DNA damage and subsequent repair activity were evaluated by staining for 8-hydroxy-deoxyguanosine (8-OHdG) and APE1, respectively; 8-OHdG immunoreactivity increased throughout the colonic mucosa, while APE1 levels in the surface epithelium increased at an earlier timepoint. Taken together, our data suggest that changes in APE1 expression after DSS treatment occurred earlier and were more widespread than changes in MMR expression, suggesting that APE1 is more sensitive for prediction of DNA deterioration in DSS-induced colitis.

Aberrant expression of apurinic-apyrimidinic endonuclease-1 (APEX1) has been reported in numerous human solid tumors and is positively correlated with cancer progression; however, the role of APEX1 in tumor progression is poorly defined. Here, we show that APEX1 contributes to aggressive colon cancer behavior and functions as an upstream activator in the Jagged1/Notch signaling pathway. APEX1 overexpression or knockdown in human colon cancer cell lines induced profound changes in malignant properties such as cell proliferation, anchorage-independent growth, migration, invasion, and angiogenesis in vitro and in tumor formation and metastasis in mouse xenograft models. These oncogenic effects of APEX1 were mediated by the upregulation of Jagged1, a major Notch ligand. Furthermore, APEX1 expression was associated with Jagged1 in various colon cancer cell lines and in tissues from colon cancer patients. This finding identifies APEX1 as a positive regulator of Jagged1/Notch activity and suggests that it is a potential therapeutic target in colon cancers that exhibit high levels of Jagged1/Notch signaling.

Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous depolarization (pacemaker potentials) responsible for the production of slow waves in gastrointestinal smooth muscle. Under current clamping, ICCs had a mean resting membrane potential of -58 ± 3 mV and externally applied ET produced membrane depolarization in a dosedependent manner. These effects were reduced by intracellular GDP beta S. A comparison of the concentration-dependent membrane depolarizations on pacemaker potentials to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1≥ET-2≥ET-3 in cultured murine small intestinal ICCs. The pretreatment with Ca(2+)-free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the ET-1 induced membrane depolarizations. Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the ET-1 induced effects on pacemaker potentials. Pretreatment with BQ-123 (ET(A )receptor antagonist) or BQ-788 (ET(B )receptor antagonist) blocked the ET-1 induced effects on pacemaker potentials in cultured murine small intestinal ICCs. However, pretreatment with BQ-788 selectively did not block the ET-1 induced effects on pacemaker potentials in cultured murine large intestinal ICCs. Also, only externally applied selective ET(B )receptor agonist, IRL 1620 did not show any influence on pacemaker potentials in cultured murine large intestine ICCs. RT-PCR results indicated the presence of the ET(A )and ET(B )receptor in ICCs. These results suggested that ET-1 modulates pacemaker potentials through ET(A )and ET(B )receptor activation in murine small intestinal ICCs and ET(A )receptor activation in murine large intestinal ICCs by external Ca(2+) influx and internal Ca(2+) release via protein kinase C or cyclooxygenase-independent mechanism. Therefore, the ICCs are targets for ET and their interaction can affect intestinal motility.

Apurinic/apyrimidinic endonuclease (APE) acts as a regulator of p53 or vice versa in the cellular response to oxidative stress. Since oxidative stress-induced apoptosis is suggested in the pathophysiology of diabetic nephropathy, we proposed that APE may have a feasible role in the progression of diabetic complications. We investigated the interrelationship between APE and p53 in streptozotocin-induced diabetic rat kidneys. Variable parameters on kidneys were checked 12 weeks after streptozotocin administration with or without chitosan oligosaccharide (COS) treatment. Streptozotocin administration caused changes as seen in early diabetic nephropathy with increased kidney size, increased p53, decreased APE, and increased cleaved caspase-3. COS was not suspected as being detrimental to renal measurements, and caused the augmentation of APE after streptozotocin administration. The augmented APE, in association with increased p53, suppressed cleaved caspase-3. 8-OHdG was mainly immunolocalized in the distal tubules, but also in the proximal tubules after streptozotocin administration without COS treatment, while APE was observed in proximal tubules in all groups. These results suggested that p53-dependent apoptosis resulting in suppressed APE might be an underlying mechanism of streptozotocin-induced nephropathy.

Cholesterol transport proteins are a prerequisite for neurosteroidogenesis. Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain-containing proteins, such as StAR and START domain-containing 6 (StarD6), are known to be distributed in the brain. Since perinatal hypothyroidism affects cerebellar development, we examined postnatal changes in StAR and StarD6 immunolocalization in the developing cerebellum of control and hypothyroid rats. Pregnant Sprague-Dawley rats were given 0.05% 6-propyl-2-thiouracil (PTU) or water from gestation day 11 until postnatal day (P) 28, and were then killed together with age-matched control rats. As shown by calbindin D-28k immunostaining, the developing cerebellar cytoarchitecture and Purkinje cells were affected by PTU-induced hypothyroidism as compared to control rats. The immunolocalization of StAR and StarD6 generally followed the maturation pattern of Purkinje cells from the vermis to the cerebellar hemisphere. StAR immunostaining first appeared in the Purkinje cells of the vermis at P7 in both control and hypothyroid rats. In control rats, a few StarD6 immunoreactive cells were seen at birth and a nuclear localization of StarD6 in Purkinje cells was obvious at P14. PTU-induced hypothyroidism delayed the appearance of StarD6 immunopositive cells until P7. Moreover, the nuclear localization of StarD6 in PTU-treated rats was not obvious at P14. An adult-like distribution of StAR and StarD6 was achieved by P21 in control and hypothyroid rats. These results suggest that StarD6 may affect the development of Purkinje cells during the first and second postnatal weeks, a known period of thyroid hormone action.

Background/Aims Heat shock proteins (HSPs) protect rats from cerulein-induced acute pancreatitis (AP) by preventing the subcellular redistribution of cathepsin B and the activation of trypsinogen. Autophagy plays a critical role in the secretion of digestive enzymes and triggering of cerulein-induced AP via the colocalization of trypsinogen and lysosomes. Therefore, using a rat cerulein-induced AP model, we investigated whether HSPs prevent AP by regulating autophagy. Methods Twelve hours after fed standard laboratory chow and water, the experimental groups (cerulein, water-immersion [WI]-cerulein and heat-shock [HS]-cerulein) and the control groups (control, WI, and HS) received one intraperitoneal injection of cerulein (50 µg/kg) or saline, respectively. All of the rats were sacrificed at 6 hours after injection. The severity of the AP was assessed based on the serum amylase level and the histological and electron microscopy findings. Western blotting was also performed for HSP60/70 and LC3B-II. Results WI and HS induced HSP60 and HSP70, respectively. The induced HSP60/70 effectively prevented the development of cerulein-induced AP. Autophagy developed in the rats with cerulein-induced AP and was documented by the expression of LC3-II and electron microscopy findings. The WI-stressed rats and HS-treated rats did not develop cerulein-induced autophagy. Conclusions HSPs exert protective effects against cerulein-induced AP in rats by inhibiting autophagy.