Involvement of human heat shock protein 90 alpha in nicotine-induced apoptosis

There have been conflicting reports of the apoptotic effects of nicotine on human cells and those studies reporting nicotine-induced apoptosis have not unequivocally clarified the molecular mechanisms underlying the effect. However, we found here that human RSa cells, established from embryonic fibroblastic cells doubly infected with Rous sarcoma virus and Simian virus 40, underwent apoptosis when cultured with medium containing 0.06-0.6 microM nicotine. The apoptosis was assessed by cellular DNA fragmentation and caspase-3 protease activation. Viability of RSa cells was reduced by nicotine treatment, as analyzed by MTT assay and the reduction was lessened by combination treatment with a caspase-3 inhibitor, acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspart-1-al (Ac-DEVD-CHO). Levels of expression of heat shock protein 90 alpha (Hsp90 alpha) were found to be increased 20 min after the nicotine treatment, as analyzed by polymerase chain reaction-based mRNA differential display after Northern blotting analysis of mRNA amounts. Cellular contents of Hsp90 alpha were furthermore increased in the nicotine-treated RSa cells, as quantitated by Western immunoblot analysis. By contrast, in RSa cells treated with nicotine in combination with geldanamycin (GA), an inhibitor of Hsp90 alpha function, DNA fragmentation was not detected and caspase-3 protease activity levels were the same as those of mock-treated cells. Nicotine-induced caspase-3 activation and Hsp90 alpha expression, as well as suppression of the induction by GA, were also observed in a xeroderma pigmentosum patient-derived cell line, XP2OS cells. Thus, it was suggested that nicotine induces apoptosis, possibly via Hsp90 alpha expression, in human cells tested.     

Heat shock protein 60 regulation of the mitochondrial permeability transition pore in tumor cells

Mitochondrial apoptosis plays a critical role in tumor maintenance and dictates the response to therapy in vivo; however, the regulators of this process are still largely elusive. Here, we show that the molecular chaperone heat shock protein 60 (Hsp60) directly associates with cyclophilin D (CypD), a component of the mitochondrial permeability transition pore. This interaction occurs in a multichaperone complex comprising Hsp60, Hsp90, and tumor necrosis factor receptor-associated protein-1, selectively assembled in tumor but not in normal mitochondria. Genetic targeting of Hsp60 by siRNA triggers CypD-dependent mitochondrial permeability transition, caspase-dependent apoptosis, and suppression of intracranial glioblastoma growth in vivo. Therefore, Hsp60 is a novel regulator of mitochondrial permeability transition, contributing to a cytoprotective chaperone network that antagonizes CypD-dependent cell death in tumors.     

鼻咽癌变过程中DNA依赖蛋白激酶表达水平研究

Objective:To observe the expression of DNA-dependent protein kinase (DNA-PKcs) in the nasopharyngeal tissues during carcinogenesis.Methods:Patients including 30 cases of nasopharyngitis,42 cases of nasopharyngeal atypical hyperplasia and 34 cases of nasopharyngeal carcinoma (NPC) were chosen from Zhongshan area in which there is high incidence rate of NPC.The expression of DNA-PKcs in the nasopharyngeal tissue was examined by immunohistochemistry.Results:The expression rates of DNA-PKcs in the nasopharyngitis tissue,the nasopharyngeal precancerous lesion and the NPC were 6.7%,64.3% and 55.9%,respectively (P＜0.001).Conclusion:The expression of DNA-dependent protein kinase may play an important role in nasopharyngeal tissue carcinogenesis.     

Competitive and noncompetitive inhibition of the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase that is involved in mammalian DNA double-strand break repair. The catalytic subunit of DNA-PK (DNA-PKcs) shares sequence homology in its kinase domain with phosphatidylinositol (PI) 3-kinase. Here, we provide a detailed kinetic analysis of DNA-PK inhibition by the PI 3-kinase inhibitor wortmannin and demonstrate this inhibition to be of a noncompetitive nature, with a Ki of 120 nM. Another inhibitor of PI 3-kinase. LY294002, its parent compound, quercetin, and other derivatives have also been studied. These chemicals are competitive inhibitors of DNA-PK, with LY294002 having a Ki of 6.0 microM. Using an antibody to wortmannin, we found that this compound binds covalently to the kinase domain of DNA-PKcs both in vitro and in vivo. Binding of wortmannin to the active site of DNA-PKcs is inhibited by ATP but not by a peptide substrate. Furthermore, wortmannin is able to bind to DNA-PKcs independently of Ku, and it is not stimulated by the presence of DNA. This suggests that the ATP binding site of DNA-PKcs is open constitutively and that DNA activation of the kinase is mediated via another mechanism.     

Targeting heat shock proteins in cancer

Heat shock proteins (HSPs) HSP27, HSP70 and HSP90 are powerful chaperones. Their expression is induced in response to a wide variety of physiological and environmental insults including anti-cancer chemotherapy, thus allowing the cell to survive to lethal conditions. Different functions of HSPs have been described to account for their cytoprotective function, including their role as molecular chaperones as they play a central role in the correct folding of misfolded proteins, but also their anti-apoptotic properties. HSPs are often overexpressed in cancer cells and this constitutive expression is necessary for cancer cells’ survival. HSPs may have oncogene-like functions and likewise mediate “non-oncogene addiction” of stressed tumor cells that must adapt to a hostile microenvironment, thereby becoming dependent for their survival on HSPs. HSP-targeting drugs have therefore emerged as potential anti-cancer agents. This review describes the different molecules and approaches being used or proposed in cancer therapy based on the in inhibition of HSP90, HSP70 and HSP27.     

Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73

Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.

Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.

Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37?°C after heat treatment at 44?°C for 15?min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3?h of incubation at 37?°C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3?h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.

Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.     

Deficiency in the catalytic subunit of DNA-dependent protein kinase causes down-regulation of ATM

Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous end-joining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.     

DNA-dependent protein kinase: a major protein involved in the cellular response to ionizing radiation

DNA-dependent protein kinase (DNA-PK) is a DNA-activated nuclear serine/threonine protein kinase. DNA-PK consists of a regulatory sub-unit, the heterodimeric Ku protein (composed of a 70- and a 86-kDa subunit) which binds DNA ends and targets the catalytic sub-unit, DNA-PKcs to DNA strand breaks. DNA-PK plays a major role in the repair of double-strand breaks induced in DNA after exposure to ionizing radiation as shown by the extreme radiosensitivity of cells with mutations in Ku86, Ku70 or DNA-PKcs genes. Cells deficient in DNA-PK activity also exhibit hypersensitivity to genotoxic drugs such as cisplatin and nitrogen mustards. In the first part of this review, the current knowledge on the biochemical characteristics of DNA-PK, its mechanism of action in DNA repair and the phenotype of DNA-PK deficient cells is summarized. These results suggest that DNA-PK might play a role in the acquisition of a resistant phenotype of human tumors to radiotherapy, chemotherapy using genotoxic drugs or to both treatments. In the second part of this review, the studies devoted to inhibition of DNA-PK in order to enhance cancer therapy by DNA-damaging agents are presented.     

DNA-dependent protein kinase in leukaemia cells and correlation with drug sensitivity

We investigated whether the levels of the DNA-dependent protein kinase (DNA-PK) activity and content correlate with drug sensitivity in different tumour materials and if this can be utilised in predicting treatment outcome. DNA-PK activity and expression were investigated in tumour cells from 8 patients with chronic lymphocytic leukaemia (CLL) and 18 patients with acute myeloid leukaemia (AML), using Western blot and DNA-PK kinase activity assay. Tumour cells from the patients were investigated for their drug sensitivity to topoisomerase II inhibitors (doxorubicin and etoposide), DNA reactive agents (melphalan, 4-hydroxycyclophosphamide and cisplatinum), an antimetabolite (cytosine arabinoside) and an antimicrotubule agent (vincristine) by fluorometric microculture cytotoxicity assay (FMCA). Within each group of leukaemia there was a large variation in both DNA-PK activity and DNA-PKcs expression, while the Ku subunits were expressed more homogeneously. In CLL cells, sensitivity to topoisomerase II inhibitors correlated with DNA-PKcs protein expression (r=0.7174, p=0.0452). In AML samples, sensitivity to DNA cross-linking alkylating agents correlated with Ku86 (r=-0.7512, p=0.0031) and Ku70 (r=-0.6134, p=0.0258) expression. Unexpectedly, DNA-PK activity was found to correlate with sensitivity to vincristine in both CLL (r=0.8557, p=0.0067) and AML (r=0.5480, p=0.0228) cells. The results indicate that DNA-PK is not only involved in the recognition of DNA double-strand breaks (DSB), but also other DNA lesions.     

Over-expression of heat shock proteins in carcinogenic endometrium

We have previously shown that the subcellular localization of beta-catenin changes according to the cell proliferation status of the human endometrium, suggesting a role of intercellular transduction in cell growth control in human endometrium not only in the physiological but also in the carcinogenic condition. To further study the possible role of heat shock proteins (HSPs) in growth control, we immunohistochemically analyzed 92 endometrial samples, 30 of normal endometrium, 20 of endometrial hyperplasia and 42 of endometrial cancer, for expression of HSP27, HSP70, HSP90, estrogen receptor (ER) and progesterone receptor. HSP27 and HSP90 were detected in endometrial epithelium strongly in the proliferative phase and weakly in the secretory phase during the menstrual cycle according to the serum estradiol level. However, they were over-expressed in endometrial hyperplasia, especially HSP27. In endometrial cancer, HSP27 expression was heterogenic among the glands and lower than that in the proliferative phase and endometrial hyperplasia. HSP27 over-expression was also observed in samples including endometrial cancer and associated hyperplasia. Results of Western blotting followed those of immunohistochemistry. HSP70 was not changed during the menstrual cycle, as HSP27 and HSP90 were, and was rather stably expressed in endometrial hyperplasia and cancer. Our results suggest that HSP27 and HSP90 contribute to cell proliferation in endometrial epithelium and that over-expression of HSP27 in endometrial hyperplasia occurs as a result of the activated condition of ER, though in cancer it decreases according to the loss of function of ER.     

低剂量放射超敏感性和放射拒抗与DNA依赖蛋白激酶的表达

低剂量放射超敏感性是最近十几年来研究的热点课题之一。是继放射敏感性之后的又一重大发现,是对传统放射生物学的一个有益补充和发展。有助于解决肿瘤细胞的放射耐受和正常组织的放射损伤问题。对放射治疗分割方法的设计、物理计划设计和生物学优化都具有重要指导意义。低剂量放射超敏感性主要包括放射超敏感期和放射拒抗期。放射超敏感期与细胞凋亡密切相关,放射拒抗期主要以DNA损伤修复后非同源末端连接相关,DNA-PK(DNA-PKcs、Ku70、Ku80)等蛋白参与DNA非同源末端连接,在超敏中具有重要作用。本文对DNA-PK在低剂量超敏感性中的作用做一综述。     

Flow cytometric measurements of heat shock proteins

A fixation and immunofluorescence staining procedure for measurements of heat shock proteins (hsp) by flow cytometry is reported. Three fixatives were compared: 80% methanol at —20°C for 1 h, 70% ethanol at 0°C for 1 h, and 3% paraformaldehyde at 4°C for 1 h followed by 0·2% NP-40. Cells fixed with methanol showed strongest immunofluorescence and lowest nonspecific fluorescence. The level of hsp 70 as a function of time after heating followed the same kinetics as the development of thermotolerance reported by others. The level of hsp 70 increased with increasing heat dose up to a maximum heat dose, and above this heat dose a decrease in the level of hsp 70 was observed. Correlated measurements of the level of hsp 70 and DNA showed that hsp 70 was found in all phases of the cell cycle. The level of hsp 70 increased about two-fold in unheated cells throughout the cell cycle. The increase in G₂ + M cells compared with G₁ cells was lower in cells heated at 45°C for 20 min followed by incubation at 37°C for 24 h before fixation and staining, than in unheated cells. The results show that flow cytometry provides a rapid and quantitative technique for measuring hsp. Correlated measurements of hsp and other cellular parameters might also be obtained.     

Role of DNA-dependent protein kinase catalytic subunit in cancer development and treatment

DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key component of the non-homologous end-joining (NHEJ) pathway, is involved in DNA double-strand break repair, immunocompetence, genomic integrity, and epidermal growth factor receptor signaling. Clinical studies indicate that expression and activity of DNA-PKcs is correlated with cancer progression and response to treatment. Various anti-DNA-PKcs strategies have been developed and tested in preclinical studies to exploit the benefit of DNA-PKcs inhibition in sensitization of radiotherapy and in combined modality therapy with other antitumor agents. In this article, we review the association between DNA-PKcs and cancer development and discuss current approaches and mechanisms for inhibition of DNA-PKcs. The future challenges are to understand how DNA-PKcs activity is correlated with cancer susceptibility and to identify those patients who would most benefit from DNA-PKcs inhibition.     

DNA依赖蛋白激酶与大肠癌淋巴转移相关性研究

Objective:To investigate DNA-dependent protein kinase(DNA-PK)expression,and its relationship with lymphatic metastasis in colorectal cancer.Methods:Tumor tissues from 60 patients,divided into two groups according to lymphatic metastasis,were immunohistochemically stained to detect the DNA-PK expression including Ku70,Ku80 and PKcs proteins.Results:Positivity of both Ku70 and Ku80 in colorectal cancer was negatively correlated with lymphatic metastasis with an rvalue of-0.57 and -0.38,respectively.Similar correlation was found between Ku expression,especially Ku70.and long-term survival.PKcs,however,displayed no significant correlation.Statistical analysis failed to detect any correlation between DNA-PK expression,and clinical characteristics,such as age,sex,tumor Jocation,tumor thickness and distant metastasis(P＞0.05).Conclusion:DNA-PK expression,especially Ku70 expression,is negatively correlated with lymphatic metastasis,and the survival of patients with colorectal cancer.Ku70 expression may be a potential indicator for the preoperative evaluation,and prognosis in colorectal cancer.