A three-dimensional tumor cell defect in activating autologous CTLs is associated with inefficient antigen presentation correlated with heat shock protein-70 down-regulation

We described previously a CTL clone able to lyse the autologous carcinoma cell line IGR-Heu after specific recognition of an HLA-A2/mutated alpha-actinin-4 peptide complex. Here, we used IGR-Heu, cultured either as standard two-dimensional monolayers or as three-dimensional spheroids, to further analyze the influence of target architecture on CTL reactivity. Interestingly, we found that changes in the tumor structure from two- to three-dimensional induced a dramatic decrease in its capacity to activate autologous CTL, as measured by IFN-gamma and tumor necrosis factor-alpha secretion. These functional alterations were attributable neither to MHC class I expression nor to tumor antigen (Ag) down-regulation, because IGR-Heu, cultured as two- or three-dimensional, expressed similar levels of HLA-A2 and alpha-actinin-4. More importantly, incubation of three-dimensional cells with synthetic epitope completely restored cytokine release by CTL. This defective Ag presentation correlated with a decrease in heat shock protein (hsp)70 expression by three-dimensional tumors compared with two-dimensional cells. Furthermore, transfection of the tumor cells with hsp70 cDNA completely restored the Ag-presenting potential of spheroids and, therefore, cytokine production by T cells. These data strongly suggest that hsp70 down-regulation in three-dimensional cells may result in tumor resistance to the immune response.     

Heat shock protein 70 (HSP70) does not prevent the inhibition of cell growth in DU-145 cells treated with TGF-beta1

BACKGROUND: Mitogen-activated protein kinase (MAPK) is one of the transforming growth factor-beta (TGF-beta signaling pathways while heat shock protein 70 (HSP70) prevents apoptosis by affecting MAPK signaling downstream. However, the interrelationship between TGF-beta and HSP70 signaling is still unknown. MATERIALS AND METHODS: DU-145 prostate cancer cells were treated with 40 pM and 200 pM TGF-beta1. After 3, 6, 9, 12 and 24 hours, cell proliferation assay and cell cycle analysis were performed. The activities of HSP70 and MAPKs (c-Jun N-terminal kinase 1 (JNK1), extracellular signal-regulated kinase 1 (ERK1), ERK2 and p38) were analyzed by Western blot at each time-point. RESULTS: TGF-beta1 inhibited the cell growth in a dose-dependent manner at 3 hours. Late G1 accumulation in the cell cycle was observed in a dose-dependent manner after 24 hours. HSP70 and JNK1 increased only at 3 hours and decreased for up to 24 hours thereafter. ERK1, ERK2 and p38 decreased from 3 to 24 hours after TGF-beta1 treatment. CONCLUSION: These data suggest that HSP70 does not prevent the inhibition of cell growth in DU-145 cells treated with TGF-beta1.     

CD70/CD27 ligand, a member of the TNF family, is expressed in human brain tumors

CD70 (CD27 ligand) has been implicated in proapoptotic signals mediated by its receptor CD27 in lymphocytes as well as in proliferative effects induced by reverse signaling in CD70-positive hematopoetic tumor cells. We were able to show that CD70 is expressed at the mRNA and protein level in human meningioma cells, glioma cells from solid human gliomas as well as glioma cell lines and 1 ependynoma. The intensity of CD70 expression varies considerably between different samples from 1 tumor type. In the U343 glioma cell line, CD70 was preferentially localized in the cell processes. Thus, our studies identify CD70 as a new marker molecule in brain tumors that are of nonhematopoetic origin.     

热休克蛋白70mRNA反义寡核苷酸对荷瘤小鼠膀胱癌的作用

目的探讨热休克蛋白70（HSP70）mRNA反义寡核苷酸（ASO）对体内膀胱癌发生发展的影响及其可能的作用机制。方法用培养的人类膀胱癌细胞株BIU-87建立40只BALB／c裸鼠膀胱癌皮下荷瘤模型，待皮下肿瘤体积约为100mm^3时，随机分为4组，每组10只。HSP70 mRNA ASO加丝裂霉素C（MMC）处理组；HSP70 mRNA反义寡核苷酸组；丝裂霉素C组；空白对照组。HSP70 mRNA按10mg／kg，肿瘤局部注射，每周2次；MMC 0．1mg／kg，腹腔注射，每周2次，空白组用生理盐水代替上述处理。治疗的第30天托颈处死裸鼠，剥离出皮下肿瘤，照像并称重量。免疫组织化学法检测肿瘤组织中的微血管密度（MVD），RT—PCR和Western blotting检测HSP70表达；原位末端标记法（TUNEL）检测细胞凋亡。结果HSP70 mRNA ASO加MMC组肿瘤抑制率超过50％，高于HSP70 mRNA ASO组或MMC组，差异有统计学意义（P〈0．05）。HSP70 mRNA ASO和MMC组相比，差异无统计学意义（P〉0．05）。ASO＋MMC组的凋亡指数（AI）分别高于其他三组，差异有统计学意义（P〈0．05）；MMC和ASO组AI分别高于空白组（P〈0．05）；MMC和ASO组相比，差异无统计学意义（P〉0．05）。肿瘤组织中的MVD与上述结果一致。结论HSP70 mRNA ASO局部注射可以抑制肿瘤的生长，其作用机制可能和其抑制微血管的形成及促进细胞凋亡有关。     

来源于人膀胱癌细胞株EJ的HSP70负载的DC所激发的特异性CTL细胞的体外应答效应

Objective: To investigate whether human dendritic cells (DC) derived from peripheral blood mononuclear cells (PBMC), which were pulsed by heat shock protein 70 (HSP70) isolated from human bladder tumor cell lines of EJ, were able to induce peptide specific cytotoxic T-lymphocytes (CTL) response in vitro and give the experimental foundation for the future clinical trials of immunotherapy in bladder tumor. Methods: The EJ-derived HSP70 co-cultured with DC from the healthy volunteers' PBMC, along with the crude lysate (the supernatant before HSP70 purification) from EJ cells were used as the experimental groups and DC not pulsed by any tumor cells antigen were the blank control. The autologous T-lymphocytes were added into the above various DC groups, and after incubation, the stimulation indexes (SI) and interferon-γ (IFN-γ) were detected to evaluate the immune activities of various DC groups. The killing effects of CTL to target cells, EJ and Hela cells, were determined with 51Cr releasing test. Results: Both DC/HSP70 and DC/the crude lysate could effectively activate CTL in vitro and kill target cells EJ. The killing effect of DC/HSP70 to EJ was much stronger than DC/the crude lysate (the supernatant before HSP70 purification) (P < 0.05). DC without any tumor cell antigens had a lower killing power to EJ. Meanwhile, DC/ HSP70 had little killing power to Hela non-relevant to bladder tumor histopathologically as compared with EJ cells (P < 0.05). Conclusion: The DC pulsed by HSP70 derived from the autologous tumor cells could induce a peptide complexes specific CTL response to tumor cells, and the CTL response induced by the DC/HSP70 was stronger, which display the basis of the possible clinical application of DC/HSP70 for bladder tumor.     

WDRPUH, a novel WD-repeat-containing protein, is highly expressed in human hepatocellular carcinoma and involved in cell proliferation

In an attempt to disclose mechanisms of hepatocarcinogenesis and discover novel target molecules for the diagnosis and treatment of hepatocellular carcinomas (HCCs), we previously analyzed expression profiles of HCC tissues by means of human cDNA microarray. Among the genes upregulated in tumor tissues compared with their nontumor counterparts, we focused on a novel gene, termed WDRPUH, and characterized its biologic function. WDRPUH encodes a predicted 620-amino acid protein containing 11 highly conserved WD40-repeat domains. Multiple-tissue Northern blot analysis revealed its specific expression in the testis among 16 normal tissues examined. Transfection of plasmids designed to express WDRPUH-specific siRNA significantly reduced its expression in HCC cells and resulted in growth suppression of transfected cells. Interestingly, we found that WDRPUH associated with HSP70, proteins of the chaperonin-containing TCP-1 (CCT1) complex, as well as BRCA2. These findings have disclosed a novel insight into hepatocarcinogenesis and suggested that WDRPUH may be a molecular target for the development of new strategies to treat HCCs.     

Heat shock protein 70 as a predictive marker for platinum-based adjuvant chemotherapy in patients with resected non-small cell lung cancer

Objectives

Although adjuvant platinum-based chemotherapy improves survival in completely resected non-small cell lung cancer (NSCLC), its effect is limited. We evaluated whether the expression of heat shock protein 70 (Hsp70) is associated with clinical outcomes in patients with completely resected NSCLC who were treated with or without adjuvant platinum-based chemotherapy.

Patients and methods

Patients who underwent curative resection for NSCLC and diagnosed as stage IIA through IIIA were included. Immunohistochemical staining for Hsp70 was performed on surgical specimens and survival rates were compared by Hsp70 expression and adjuvant platinum-based chemotherapy.

Results

Of 327 enrolled patients, Hsp70 expression was positive in 220 (67.3%). For patients who did not receive adjuvant chemotherapy, Hsp70 expression did not significantly affect survival. However, for patients who received adjuvant chemotherapy, those with Hsp70-positive tumors had a longer disease-free survival outcome than cases with Hsp70-negative tumors (not reached vs. 27.3 months; P = 0.002), although there was no significant difference in overall survival (97.0 vs. 58.9 months, P = 0.080). In the adjuvant chemotherapy group, multivariate modeling showed that patients with Hsp70-postitive tumors had a lower risk of recurrence and death after adjusting for age, sex, performance status, pathologic stage, and histological type (disease-free survival: adjusted hazard ratio, 0.537; 95% CI, 0.362–0.796; P = 0.002; overall survival: adjusted hazard ratio, 0.663; 95% CI, 0.419–1.051; P = 0.080).

Conclusion

Hsp70 is a positive predictive factor in completely resected NSCLC with received platinum-based adjuvant chemotherapy.     

Co-overexpression of Bag-1 and heat shock protein 70 in human epidermal squamous cell carcinoma: Bag-1-mediated resistance to 5-fluorouracil-induced apoptosis

Background:

The aim was to determine whether Bcl-2-associated athanogene-1 (Bag-1) and/or its binding protein heat shock protein-70 (Hsp70) exhibit deregulated expression in epidermal squamous cell carcinoma (SCC) and whether Bag-1 confers apoptosis resistance.

Method:

Immunohistochemistry for Bag-1 and Hsp70 was performed on 60 epidermal SCC and 10 normal skin samples. The epidermal SCC cell line SCC-13 was treated with 5-fluorouracil (5-FU) after Bag-1 knockdown to determine whether high Bag-1 levels contribute to growth and/or apoptosis resistance.

Results:

Normal epithelium expressed primarily nuclear Bag-1. Most tumours showed reduced nuclear Bag-1 staining, but a subset exhibited strong Bag-1 staining, with cytoplasmic Bag-1 staining intensity correlating with cytoplasmic Hsp70 staining intensity (r_s=0.462; P<0.001) and less differentiation (P<0.001). Bag-1 knockdown resulted in markedly reduced SCC-13 cell yield, increased spontaneous apoptosis and enhanced sensitivity to 5-FU-induced apoptosis. Apoptosis induced by 5-FU in the Bag-1-knockdown cells was significantly greater than the additive apoptotic effect of 5-FU or Bag-1 knockdown alone.

Conclusions:

Overexpression of Bag-1 and Hsp70 in poorly differentiated SCC may confer both enhanced tumour cell growth and apoptosis resistance. Bag-1 may contribute to the resistance of more advanced epidermal SCC to chemotherapy.     

Enhanced T-cell immunity induced by dendritic cells with phagocytosis of heat shock protein 70 gene-transfected tumor cells in early phase of apoptosis

The dual role of heat shock protein 70 (HSP70), as antigenic peptide chaperone and danger signal, makes it especially important in dendritic cell (DC)-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T-cell stimulation and vaccine efficacy. We found that DCs with phagocytosis of MCA/HSP in early phase of apoptosis expressed more pMHC I complexes, stimulated stronger cytotoxic T lymphocyte (CTL) responses (40% specific killing at an E:T cell ratio of 50) and induced immune protection in 90% of mice against MCA tumor cell challenge, compared with 25% specific CTL killing activity and 60% immune protection seen in mice immunized with DC with phagocytosis of MCA/HSP in late phase of apoptosis (P<0.05). Similar results were confirmed in another EG7 tumor model also expressing HSP70. Taken together, our data demonstrate that HSP70 on apoptotic tumor cells stimulate DC maturation, and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DCs with phagocytosis of apoptotic tumor cells in late phase of apoptosis. These results may have an important impact in designing DC-based antitumor vaccines.     

Overexpression of the aldo-keto reductase family protein AKR1B10 is highly correlated with smokers' non-small cell lung carcinomas.

PURPOSE: Squamous cell carcinoma (SCC) and adenocarcinoma of the lung are currently subject to similar treatment regimens despite distinct differences in histology and epidemiology. The aim of this study is to identify a molecular target with diagnostic and therapeutic values for SCC. EXPERIMENTAL DESIGN: Genes specifically up-regulated in SCC were explored through microarray analysis of 5 SCCs, 5 adenocarcinomas, 10 small cell lung carcinomas, 27 normal tissues, and 40 cancer cell lines. Clinical usefulness of these genes was subsequently examined mainly by immunohistochemical analysis. RESULTS: Seven genes, including aldo-keto reductase family 1, member B10 (AKR1B10), were identified as SCC-specific genes. AKR1B10 was further examined by immunohistochemical analysis of 101 non-small cell lung carcinomas (NSCLC) and its overexpression was observed in 27 of 32 (84.4%) SCCs and 19 of 65 (29.2%) adenocarcinomas. Multiple regression analysis showed that smoking was an independent variable responsible for AKR1B10 overexpression in NSCLCs (P < 0.01) and adenocarcinomas (P < 0.01). AKR1B10 staining was occasionally observed even in squamous metaplasia, a precancerous lesion of SCC. CONCLUSION: AKR1B10 was overexpressed in most cases with SCC, which is closely associated with smoking, and many adenocarcinoma cases of smokers. These results suggest that AKR1B10 is a potential diagnostic marker specific to smokers' NSCLCs and might be involved in tobacco-related carcinogenesis.     

Aberrant over-expression of a forkhead family member, FOXO1A, in a brain tumor cell line

Background

Gefitinib, a small molecule tyrosine kinase inhibitor of the Epidermal Growth Factor Receptor (EGFR), has shown limited efficacy in the treatment of lung cancer. Recognized clinical predictors of response to this drug, specifically female, non-smoker, Asian descent, and adenocarcinoma, together suggest a genetic basis for drug response. Recent studies have addressed the relationship between response and either sequence mutations or increased copy number of specific receptor tyrosine kinases. We set out to examine the relationship between response and the molecular status of two such kinases, EGFR and HER2, in 39 patients treated with gefitinib at the BC Cancer Agency.

Methods

Archival patient material was reviewed by a pathologist and malignant cells were selectively isolated by laser microdissection or manual recovery of cells from microscope slides. Genomic DNA was extracted from 37 such patient samples and exons 18–24, coding for the tyrosine kinase domain of EGFR, were amplified by PCR and sequenced. EGFR and HER2 copy number status were also assessed using FISH in 26 samples. Correlations between molecular features and drug response were assessed using the two-sided Fisher's exact test.

Results

Mutations previously correlated with response were detected in five tumours, four with exon 19 deletions and one with an exon 21 missense L858R point mutation. Increased gene copy number was observed in thirteen tumours, seven with EGFR amplification, three with HER2 amplification, and three with amplification of both genes. In our study cohort, a correlation was not observed between response and EGFR mutations (exon 19 deletion p = 0.0889, we observed a single exon 21 mutation in a non-responder) or increases in EGFR or HER2 copy number (p = 0.552 and 0.437, respectively).

Conclusion

Neither mutation of EGFR nor increased copy number of EGFR or HER2 was diagnostic of response to gefitinib in this cohort. However, validation of these features in a larger sample set is appropriate. Identification of additional predictive biomarkers beyond EGFR status may be necessary to accurately predict treatment outcome.     

Heat shock protein 27 was up-regulated in cisplatin resistant human ovarian tumor cell line and associated with the cisplatin resistance

To understand the molecular basis for failure of cisplatin (CDDP) based chemotherapy, we compared gene expressions between CDDP sensitive and resistant ovarian tumor cell line, 2008 and 2008/C13*5.25, by mRNA differential display. We detected both up-regulated and down-regulated bands in the resistant cell and found some of them to be positive on Northern blotting. DNA sequencing revealed one to be mitochondrial heat shock protein 75. We found that HSP27 and HSP70 were also up-regulated in the resistant cell by Western blotting. Further, transient transfection with the HSP27 sense gene made the sensitive cell more resistant, while transient transfection with the antisense gene made it more sensitive.     

EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle

The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse redistribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells.     

BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines

Human BRG1 is a component of the evolutionarily conserved SWI-SNF chromatin remodeling complex. BRG1 has been implicated in growth control through its interaction with the tumor suppressor pRb and may consequently serve as a negative regulator of proliferation. Postulating that BRG1 may itself be a tumor suppressor gene, we screened a panel of tumor cell lines to determine whether the gene is targeted for mutation. We report that the COOH-terminal region of BRG1 is homozygously deleted in two carcinoma cell lines, prostate TSU-Pr1 and lung A-427. In addition, biallelic inactivations of BRG1 were observed in four other cell lines derived from carcinomas of the breast, lung, pancreas, and prostate; their mutations in BRG1 included three frameshift lesions and one nonsense lesion. Point mutations were also discovered in a number of other cell lines, however in most cases any effect of these mutations on BRG1 function remains to be established. A variety of different mutations within BRG1, in several cell lines, suggest that BRG1 may be targeted for disruption in human tumors. Significantly, reintroduction of BRG1 into cells lacking BRG1 expression was sufficient to reverse their transformed phenotype inducing growth arrest and a flattened morphology. These data strongly support the model that BRG1 may function as a tumor suppressor and strengthen the hypothesis that the regulation of gene expression through chromatin remodeling is critical for cancer progression. It will be important to confirm these observations in primary tumors.     

HSP70ASODN转染增强顺铂抑制肝癌细胞HepG2增殖的试验研究

目的:研究HSP70ASODN联合顺铂对于肝癌细胞的增殖抑制作用。方法:运用MTT法比较转染组和未转染组联合顺铂在肝癌细胞增殖方面的差异。结果:观察转染HSP70组与未转染组HepG2细胞株对化疗药物顺铂的敏感性时,24h、48h、72h、96h未转染组与转染组的细胞生长抑制率分别为,0.329±0.029、0.583±0.022;0.356±0.035、0.684±0.052;0.497±0.033、0.723±0.048;0.523±0.035、0.715±0.047。结论:HSP70ASODN能增强顺铂对HepG2增殖抑制作用。     

STI571 (Glivec) inhibits the interaction between c-KIT and heat shock protein 90 of the gastrointestinal stromal tumor cell line, GIST-T1

The gastrointestinal stromal tumor cell line, GIST-T1, has a heterogenic 57-base pair deletion in exon 11 of the c-kit mutation, and the c-KIT protein in the GIST-T1 cells constitutively activated. We report that STI571 (Glivec; Novartis, Basel, Switzerland), a specific inhibitor of c-KIT, inhibits the clustering of c-KIT at the cell membrane of the GIST-T1 cells. Furthermore, STI571 prevents the interaction between c-KIT and the molecular chaperone, heat shock protein 90 (Hsp90). Geldanamycin, an inhibitor of Hsp90, also prevents interaction between c-KIT and Hsp90, and inhibits tyrosine phosphorylation of c-KIT. Our results indicate that c-KIT molecules are assembled on the cell surface of the GIST-T1 cells, and that the interaction between c-KIT and Hsp90 plays an important role in c-KIT activation.