Atsuko Kanzaki

Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair

While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

Expression of multidrug resistance-related transporters in human breast carcinoma

The expression levels of mRNA for multidrug resistance 1 (MDR1) gene, multidrug resistance protein 1 (MRP1), lung resistance‐related protein (LRP) and breast cancer resistance protein (BCRP), which confer multidrug resistance in vitro, were examined in 43 untreated breast carcinoma patients, of whom 38 subsequently received doxorubicin‐based chemotherapy after surgery, in order to elucidate the roles of these genes in drug resistance in vivo. The mRNA levels were determined using a semi‐quantitative reverse‐transcription polymerase chain reaction method in breast carcinoma tissues including at least 80% carcinoma cells. The expression level of BCRP gene was low and did not vary markedly in comparison with that of MDR1, MRP1 or LRP gene. The expressions of MDR1 and MRP1 genes were correlated with each other, but the expression of BCRP or LRP gene did not correlate with that of other genes. These four gene expressions were independent of age, TNM categories and the status of progesterone or estrogen receptor. The expression levels of these four genes were not related to the relapse or prognosis of the 38 patients treated with doxorubicin‐based chemotherapy. P‐glycoprotein (P‐gp)/MDRl, MRP1 and LRP may play more important roles than BCRP in chemotherapy of human breast carcinoma.

Prognostic Value of the Cu-Transporting ATPase in Ovarian Carcinoma Patients Receiving Cisplatin-Based Chemotherapy

Purpose: A major obstacle in the treatment of ovarian carcinoma is the intrinsic/acquired resistance to cisplatin-based chemotherapy. Cu-transporting ATPase (ATP7B) has been reported to be associated with cisplatin resistance in vitro. However, the clinical significance of this transporter has not previously been addressed. Our goal was to investigate ATP7B expression in ovarian carcinoma and whether its expression correlates with prognosis and reduced responsiveness to cisplatin treatment. Experimental Design: We retrospectively examined the expression of ATP7B and p53 in primary ovarian carcinoma and its association with chemotherapeutic effect. Tissues were surgically removed from 104 ovarian carcinomas patients who received cisplatin-based chemotherapy. We performed immunohistochemical analysis of ATP7B and p53 using a monoclonal antibody against ATP7B and DO7 antibody against p53 protein in 104 ovarian carcinomas and adjacent nonneoplastic tissues. The significance of ATP7B and p53 in the prognosis of patients with ovarian carcinomas was also examined in the survival analysis of mortality follow-up data covering the period between 1988 and 2001. Furthermore, mutation analysis at the six Cu-binding domain and ATP-binding domain, which may be important for cisplatin transport, were performed using single-strand conformational polymorphism after reverse transcriptase-PCR. Results: A variable degree of cytoplasmic staining of ATP7B in tumor cells was observed in 34.6% (36 of 104 cases) of the analyzed carcinomas. ATP7B expression was not observed in adjacent nonneoplastic tissues. ATP7B positivity in poorly/moderately differentiated carcinoma was significantly higher than that in low malignant potential tumor/well-differentiated carcinoma (P = 0.0276). Patients with ATP7B-positive tumors had a significantly inferior response to chemotherapy compared with the patients with ATP7B-negative tumors (P = 0.025). The multivariate Cox regression analysis revealed that ATP7B expression (hazard ratio, 1.8; 95% confidence interval, 1.0–3.2, P = 0.048), as well as International Federation of Gynecologists and Obstetricians stage (hazard ratio, 2.0; 95% confidence interval, 1.1–3.6, P = 0.018), was prognostic for poor disease outcome after adjustment for p53 expression, grade, and residual tumor. p53 expression was detected in 31.5% (26/104 cases). No mutation was observed on the six Cu-binding domain or ATP-binding domain in human ovarian carcinomas expressing ATP7B gene. Conclusions: This study demonstrates that overexpression of ATP7B in ovarian carcinoma is correlated with unfavorable clinical outcome in patients treated with cisplatin-based chemotherapy. Therefore, ATP7B expression may be considered as a predictive marker of chemoresistance for cisplatin-based chemotherapy in patients with ovarian carcinoma. We further predict that drugs targeting ATP7B might be useful in combination with cisplatin-based regimen for the improvement of patients with ovarian carcinoma.

Hypoxia-inducible factor 1 alpha (HIF-1α) gene expression in human ovarian carcinoma

Abstract Hypoxia-inducible factor 1α (HIF-1α) that regulates genes involved in response to hypoxia and promotes neo-angiogenesis, is a transcriptional factor for vascular endothelial cell growth factor (VEGF). The aim of this study was to examine the expression of HIF-1α and VEGF gene expressions and their relation to angiogenesis, clinicopathologic variables and survival in the patient with human ovarian carcinoma. We retrospectively analyzed HIF-1α and VEGF gene expression levels using reverse transcriptase polymerase chain reaction (RT-PCR) in 60 ovarian carcinomas. Intratumoral microvessel density (IMD) was assessed by immunostaining endothelial cells, using anti-CD 31 antibody in frozen sections. The relationships between the expression level of these genes, IMD and clinicopathologic variables were evaluated by Students t-test and chi-square tests. Survival analysis was performed by Kaplan–Meier curves. HIF-1α or VEGF gene expression level was independent of age, clinical stage and histological subtype besides grade of tumor. There was no relationship between HIF-1α or VEGF gene expression level and IMD in all carcinomas (R=0.118 and 0.224, respectively). In addition, a weak association between HIF-1α and VEGF gene expression level was observed (R=0.300, P=0.020). The association between VEGF gene expression and IMD was observed (R=0.501, P=0.016). However, no association between IMD and HIF-1α gene expression was observed. Further, both HIF-1α and VEGF gene expression levels had no effect on survival in the patient with ovarian carcinoma. These results suggest that VEGF upregulated by HIF-1α gene may be involved in angiogenesis of some type of ovarian carcinoma, but the expression levels of both genes have no effect on survival in the patients with ovarian carcinoma.

Loss of heterozygosity of nucleotide excision repair factors in sporadic ovarian, colon and lung carcinomas: implication for their roles of carcinogenesis in human solid tumors

The deficiencies of nucleotide excision repair (NER) factors are genetic diseases, xeroderma pigmentosum (XP) increasing risk of developing cancer on sun-exposed areas of the skin. However, the abnormality of NER factors in human sporadic carcinoma remains unclear. Loss of heterozygosity (LOH) analysis for the XP, XPA, XPB, XPC, XPD, XPE, XPF, XPG and the transcription-coupled repair factor, Cockayne syndrome B (CSB) revealed that NER factors were abnormal in 62.1 % of ovarian tumors (18/29), 16.7% of colon (2/12) and 22.2% lung (2/9) carcinomas. Furthermore, 13.8% of ovarian, 8.3% of colon and 22% of lung carcinomas exhibited LOH for NER factors without LOH for tumor suppressor genes such as p53, FHIT, APC, BRCAI, BRCA2 and DCC. Although both microsatellite instability and LOH of NER factors were observed in some cases, there was no strong association between them in the present study. These observations raise the possibility that alterations of NER factors may be frequent in human sporadic carcinomas. Further study should be needed to find the direct evidence of NER gene abnormalities in human sporadic carcinoma tissues.

Expression of copper-transporting P-type adenosine triphosphatase (ATP7B) as a chemoresistance marker in human oral squamous cell carcinoma treated with cisplatin

An important clinical problem in the treatment of oral squamous cell carcinoma is the intrinsic/acquired resistance to cisplatin-based chemotherapy. Copper-transporting P-type adenosine triphosphate (ATP7B) has been reported to be associated with cisplatin resistance in vitro (Komatsu et al., Cancer Res 60, 1312-1316,2000). However, the clinical significance of this transporter has not previously been addressed. Our aim of this study was to investigate if ATP7B is expressed in oral squamous cell carcinoma and whether its expression correlates with prognosis and reduced responsiveness to cisplatin treatment. Biopsy tissues were obtained from the tumors of 70 patients with oral SCC, and 51 patients received cisplatin-based preoperative chemotherapy. We performed immunohistochemical analysis of ATP7B using monoclonal antibody against ATP7B in 51 oral SCC and adjacent neoplastic tissues. The significance of ATP7B in the prognosis of patients with oral SCC was also examined in the survival analysis of mortality follow-up data covering the period 1991 through 2000. We retrospectively examined the expression of ATP7B in primary oral SCC carcinoma and its association with chemotherapeutic effect. A variable degree of cytoplasmic staining of tumor cells was observed in 54.9% (28/51 cases) of the analyzed carcinomas. Patients with ATP7B-positive tumors had a significantly inferior response to chemotherapy compared with the patients with ATP7B-negative tumors (P = 0.03). The patients who received cisplatin-based chemotherapy with ATP7B-positive carcinomas had a significantly poorer overall survival than those with ATP7B-negative tumors (P = 0.015). These findings suggest that high levels of ATP7B expression in oral SCC are associated with unfavorable clinical outcome in patients with oral SCCs treated with cisplatin-based chemotherapy. ATP7B expression may be a preoperative indicator for a choice of cisplatin in some patients.

Copper‐transporting P‐Type Adenosine Triphosphatase (ATP7B) Is Expressed in Human Breast Carcinoma

This is the first report to show that a copper‐transporting P‐type adenosine triphosphatase, ATP7B, is expressed in certain breast carcinomas, and a priori knowledge of its expression is important for the choice of therapy. We investigated the hypothesis that ATP7B, which was shown to be associated with cisplatin resistance in vitro, is expressed in certain breast carcinomas. To test this hypothesis, ATP7B expression and protein level were examined in 41 breast carcinomas using RT‐PCR and immunohistochemistry. ATP7B gene/protein could be detected in 22.0% (9/41) of breast carcinomas and ATP7B gene expression was correlated well with the protein expression. In nine ATP7B‐positive tumors, adjacent normal breast tissue was similarly analyzed, revealing that ATP7B is upregulated in breast carcinoma. ATP7B gene expression in poorly differentiated carcinoma was significantly higher than that in well‐/moderately differentiated carcinoma (P=0.012). Furthermore, we found no association between the ATP7B gene/protein expression and that of MDR1, MRP1, LRP and BCRP. These findings suggested that ATP7B gene expression might be a chemoresistance marker for cisplatin in patients with poorly differentiated breast carcinoma.

Expression of uridine and thymidine phosphorylase genes in human breast carcinoma

Uridine phosphorylase (UPase) and an angiogenic enzyme, thymidine phosphorylase (dThdPase) are involved in degradation of the pyrimidine nucleosides through phosphorolysis. The expression levels of UPase and dThdPase are higher in human solid tumors including breast carcinomas than in normal tissues. To clarify the correlation between the expression levels of UPase and dThdPase genes and the clinicopathological factors, mRNA levels of these enzymes were examined by RT‐PCR in 43 breast carcinomas. UPase gene expression was not correlated with dThdPase gene expression (regression coefficient R = 0.032). Although the expression level of the dThdPase gene was correlated with angiogenesis, detected by immunostaining endothelial cells (R = 0.66), that of UPase gene was not (R = 0.044). These results suggest that UPase does not have a strong angiogenic activity. The UPase gene expression levels in tumors of patients who relapsed were significantly higher than in those from patients who did not (p = 0.039). Although the expression levels of neither UPase or dThdPase were associated with age, pT, pN, pM, estrogen or progesterone receptor positivity, the patients with the higher levels of UPase gene expression had worse survival (p = 0.0038) than those with lower levels. In contrast, the expression of dThdPase gene was not related to relapse or survival of these patients with breast carcinoma. Our findings suggest that the expression level of UPase gene may be an independent prognostic marker in human breast carcinoma.

Copper-transporting P-type adenosine triphosphatase (ATP7B) is expressed in human gastric carcinoma

This study describes the first report that a copper-transporting P-type adenosine triphosphatase, ATP7B, is expressed in human gastric carcinomas. Herein, we investigated the hypothesis that ATP7B, which was shown to be associated with cisplatin resistance in vitro, is expressed in certain gastric carcinomas. To test this hypothesis, ATP7B expression level was examined in 51 gastric carcinomas by immunohistochemistry. ATP7B protein could be detected in 41.2% (21/51) of gastric carcinoma by immunohistochemical analysis. In ATP7B-positive tumors, adjacent non-neoplastic tissue was similarly analyzed, revealing that ATP7B is upregulated in gastric carcinoma. ATP7B expression in poorly differentiated/undifferentiated carcinoma was significantly higher than that in well/moderately-differentiated carcinoma (P=0.0278). These findings suggested that ATP7B expression might be a chemoresistance marker against cisplatin in some patients with poorly differentiated/undifferentiated gastric carcinoma.

Different features of angiogenesis between ovarian and breast carcinoma

Angiogenesis assessed by immunohistochemical staining for endothelial cells has been widely accepted as an independent prognostic factor in human breast carcinoma. However, the clinicopathologic significance of angiogenesis is still being argued in ovarian carcinoma. Therefore, we retrospectively analyzed the clinicopathologic significance of angiogenesis in ovarian carcinoma compared with that in breast carcinoma. After vessels were stained with CD34-monoclonal antibody, the areas with the highest number of intratumoral microvessels were assessed in a 200x field in 42 ovarian carcinoma and 41 breast carcinoma. Intratumoral microvessel density (IMD) in ovarian carcinoma was significantly lower than that in breast carcinoma. Further, the difference of IMD from tumor to tumor in ovarian carcinoma was smaller than that in breast carcinoma. IMD was correlated with tumor grade, but not with other clinicopathologic variables in ovarian carcinoma. Although the patients with high-IMD tumor revealed a poorer prognosis than those with low-IMD tumor in breast carcinoma, IMD had no influential effects on the survival of the patients with ovarian carcinoma. Our comparative analysis of IMD in ovarian carcinoma with that in breast carcinoma indicates that angiogenesis may play an important role in the transient of ovarian neoplasms, but not in the progression of ovarian carcinomas, and that the biological roles of angiogenesis might be different, depending on histologic subtype.