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ABSTRACT: Ovarian clear cell carcinoma (CCC) carries a poor prognosis because of its insensitivity to chemotherapy. We previously found an association between reduced proliferation of CCC and chemoresistance; here we investigated the mechanism of the reduced proliferation.
We assessed cell cycle function by measuring the activity of cyclin-dependent kinases (CDKs) and the protein expression of cyclins, the CDK inhibitors, and p53 in 22 ovarian cancer cell lines and 60 human ovarian cancer specimens. We examined the cellular location of p27, p27 phosphorylated at threonine 157 (p27(Thr157)), and CDK2 protein by confocal microscopy and western blotting. We tested the effect of the inhibitor of phosphatidylinositol-3-kinase (PI3K) and small interfering RNA against p27 (si-p27) in two CCC cell lines (RMG-I, SMOV-2).
CCC cells had lower CDK2 activity and higher p27 expression than serous adenocarcinoma (SA) cells. Low CDK2 activity correlated with high p27 protein expression. p27(Thr157) sequestered CDK2 in the cytoplasm, but PI3K inhibitor or si-p27 maintained CDK2 in the nucleus and restored its activity. In human specimens, CDK2 was mostly in the cytoplasm and was spatially associated with p27; CDK2 activity was lower in the CCC than in the SA specimens. si-p27 enhanced the cytotoxic effect of cisplatin, doxorubicin, and gemcitabine in both RMG-I cells and SMOV-2 cells.
Reduced CDK2 activity via the cytoplasmic sequestration of CDK2 by p27(Thr157) may contribute to suppression of CCC proliferation. A prospective study is needed to determine whether the cytoplasmic sequestration of CDK2 results in the chemoresistance of CCC.
Gynecologic Oncology 06/2011; 122(3):641-7. · 3.89 Impact Factor
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Junichi Kurebayashi,
Naoki Kanomata,
Yuji Kozuka,
Takuya Moriya,
Norihiro Kikukawa,
Yuko Kawasaki,
Shigeyoshi Harada,
Shigeyuki Tamura,
Satoshi Nakayama, Hideki Ishihara,
Shinzaburo Noguchi,
Hiroshi Sonoo
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ABSTRACT: The cell cycle profile test is suggested to be an independent prognostic indicator for breast cancer patients. To further clarify the prognostic value, we applied this to breast cancer patients treated with postoperative 5-fluorouracil-based chemotherapy.
A total of 153 breast cancer patients, who were treated with postoperative 5-fluorouracil-based chemotherapies, were randomly selected. Specific activities of cyclin-dependent kinases 1 and 2 in the tumor samples were analyzed. Patients were divided into three categories (low, intermediate or high risk) based on cell cycle profile analysis.
The proportions of the cell cycle profile categories were 39% for low risk, 10% for intermediate risk and 45% for high risk, respectively. Although the cell cycle profile test did not show a significant predictive power for relapse-free survival (high vs. low risk; P = 0.052), the cell cycle profile categories were significant prognostic factors in a subgroup of 98 patients with fewer than three involved nodes (high vs. low risk, P = 0.004). Multivariate analyses also indicated that a cell cycle profile parameter (high vs. low risk) was an independent prognostic indicator from the number of involved nodes and clinical stage in this subgroup (hazard ratio = 2.46, P = 0.01). Interestingly, the prognostic power of the cell cycle profile test was significant in 75 patients treated with oral 5-fluorouracil derivatives alone (hazard ratio = 6.29 for high vs. low risk, P = 0.02).
These findings suggest that the cell cycle profile test is useful for predicting a higher risk of relapse in patients treated with postoperative 5-fluorouracil-based chemotherapy.
Japanese Journal of Clinical Oncology 06/2011; 41(6):739-46. · 1.78 Impact Factor
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ABSTRACT: Aberrant promoter methylation of genes is a common molecular event in breast cancer. Thus, DNA methylation analysis is expected to be a new tool for cancer diagnosis. In this article, we have established a new, high-performance DNA methylation assay, the one-step methylation-specific polymerase chain reaction (OS-MSP) assay, which is optimized for analyzing gene methylation in serum DNA. The OS-MSP assay is designed to detect aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 genes in serum DNA. Moreover, two quality control markers were designed for monitoring the bisulfite conversion efficiency and measuring the DNA content in the serum. Serum samples were collected from patients with primary (n = 101, stages I-III) and metastatic breast cancers (n = 58) as well as from healthy controls (n = 87). If methylation of at least one of the three genes was observed, the OS-MSP assay was considered positive. The sensitivity of this assay was significantly higher than that of the assay involving conventional tumor markers (CEA and/or CA15-3) for stages I (24 vs. 8%) and II (26 vs. 8%) breast cancer and similar to that of the assay involving the conventional tumor markers for stage III (18 vs. 19%) and metastatic breast cancers (55 vs. 59%). The results of the OS-MSP assay and those of the assay involving CEA and/or CA15-3 seemed to compensate for each other because sensitivity of these assays increased to 78% when used in combination for metastatic breast cancer. In conclusion, we have developed a new OS-MSP assay with improved sensitivity and convenience; thus, this assay is more suitable for detecting aberrant promoter methylation in serum DNA. Moreover, the combination of the OS-MSP assay and the assay involving CEA and/or CA15-3 is promising for enhancing the sensitivity of diagnosis of metastatic breast cancer.
Breast Cancer Research and Treatment 05/2011; 132(1):165-73. · 4.43 Impact Factor
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Satoshi Nakayama,
Yasuhiro Torikoshi,
Takeshi Takahashi,
Tomokazu Yoshida,
Tamotsu Sudo,
Tomoko Matsushima,
Yuko Kawasaki,
Aya Katayama,
Keigo Gohda,
Gabriel N Hortobagyi,
Shinzaburo Noguchi,
Toshiyuki Sakai, Hideki Ishihara,
Naoto T Ueno
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ABSTRACT: Paclitaxel is used widely in the treatment of breast cancer. Not all tumors respond to this drug, however, and the characteristics that distinguish resistant tumors from sensitive tumors are not well defined. Activation of the spindle assembly checkpoint is required for paclitaxel-induced cell death. We hypothesized that cyclin-dependent kinase (CDK) 1 activity and CDK2 activity in cancer cells, which reflect the activation state of the spindle assembly checkpoint and the growth state, respectively, predict sensitivity to paclitaxel.
Cell viability assays and DNA and chromatin morphology analyses were performed in human breast cancer cell lines to evaluate sensitivity to paclitaxel and the cell cycle response to paclitaxel. We then examined the specific activities of CDK1 and CDK2 in these cell lines and in xenograft models of human breast cancer before and after paclitaxel treatment. Protein expression and kinase activity of CDKs and cyclins were analyzed using a newly developed assay system.
In the cell lines, biological response to paclitaxel in vitro did not accurately predict sensitivity to paclitaxel in vivo. Among the breast cancer xenograft tumors, however, tumors with significantly increased CDK1 specific activity after paclitaxel treatment were sensitive to paclitaxel in vivo, whereas tumors without such an increase were resistant to paclitaxel in vivo. Baseline CDK2 specific activity was higher in tumors that were sensitive to paclitaxel than in tumors that were resistant to paclitaxel.
The change in CDK1 specific activity of xenograft tumors after paclitaxel treatment and the CDK2 specific activity before paclitaxel treatment are both associated with the drug sensitivity in vivo. Analysis of cyclin-dependent kinase activity in the clinical setting could be a powerful approach for predicting paclitaxel sensitivity.
Breast cancer research: BCR 03/2009; 11(1):R12. · 5.24 Impact Factor
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ABSTRACT: Cathepsin K is known to play an important role in bone resorption, and it has the P2 specificity for proline. Rat cathepsin K has 88% identity with the human enzyme. However, it has been reported that its enzymatic activity for a Cbz-Leu-Arg-MCA substrate is lower than that of human cathepsin K, and that the rat enzyme is not well inhibited by human cathepsin K inhibitors. For this study, we prepared recombinant enzyme to investigate the substrate specificity of rat cathepsin K. Cleavage experiments using the fragment of type I collagen and peptidic libraries demonstrated that rat cathepsin K preferentially hydrolyses the substrates at the P2 Hyp position. Comparison of the S2 site between rat and human cathepsin K sequences indicated that two S2 residues at Ser134 and Val160 in rat are varied to Ala and Leu, respectively, in the human enzyme. Cleavage experiments using two single mutants, S134A and V160L, and one double mutant, S134A/V160L, of rat cathepsin K showed that all the rat mutants lost the P2 Hyp specificity. The information obtained from our comparative studies on rat and human cathepsin K should make a significant impact on developing specific inhibitors of human cathepsin K since rat is usually used as test species.
Journal of Biochemistry 08/2008; 144(4):499-506. · 2.37 Impact Factor
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ABSTRACT: Adiponectin is one of the most important adipocytokines secreted from adipose tissue. In addition to its effects on glucose and fatty acid metabolism, it has been reported that adiponectin has a direct growth-inhibitory effect on breast cancer cells. However, it still remains to be established how adiponectin affects cell cycle and apoptosis and whether or not its inhibitory effect is mediated through adiponectin receptors. Here, we demonstrated that adiponectin treatment resulted in a significant dose-dependent growth inhibition of both MDA-MB-231 and T47D cells. In both cell lines, the G0/G1 population significantly increased after adiponectin treatment, but apoptosis was not induced. High expression of mRNA and protein of adiponectin receptor 1 was observed, but expression of adiponectin receptor 2 was very low in both cell lines. Treatment with small interference RNA against adiponectin receptor 1 significantly reduced the growth inhibition induced by adiponectin in both cell lines. Taken together, adiponectin decreases breast cancer cell proliferation by inhibiting the entry into S-phase without inducing apoptosis, and this inhibitory effect is mediated through adiponectin receptor 1.
Breast Cancer Research and Treatment 01/2008; 112(3):405-10. · 4.43 Impact Factor
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ABSTRACT: The present study investigated the mRNA expression level of ubiquitin c-terminal hydrolase (UCH)-L1 and -L3 in breast cancer tissue and aimed to elucidate its association with tumor characteristics and patient prognosis. UCH-L1 and UCH-L3 mRNA levels in invasive breast cancer (n = 100) were determined by a real-time polymerase chain reaction (PCR) assay and their relationship with various clinicopathological characteristics of breast tumors as well as patient prognosis were studied. UCH-L3 mRNA level was significantly upregulated in breast cancer tissue compared to adjacent normal breast tissue (P < 0.005), and UHC-L1 mRNA level also showed a non-significant increase in tumor tissue compared to adjacent normal breast tissue. Both UCH-L1 and UCH-L3 mRNA levels were significantly higher in high histological grade tumors than in low histological grade tumors (P < 0.001 and P < 0.005, respectively). High UCH-L1 mRNA level was significantly associated with negative estrogen receptor status (P < 0.05) and negative progesterone receptor status (P < 0.05). Patients with both UCH-L1 and UCH-L3 mRNA high tumors showed a significantly poorer prognosis than those in the UCH-L1 or UCH-L3 mRNA low group (P < 0.005). These observations that UCH-L3 mRNA level is upregulated in breast cancer tissue, and breast tumors with both UCH-L1 and UCH-L3 mRNA high expression are associated with a poor prognosis, suggest the possible involvement of UCH-L1 and UCH-L3 in the pathogenesis and progression of breast cancer.
Cancer Science 06/2006; 97(6):523-9. · 3.33 Impact Factor
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Hideki Ishihara,
Tomokazu Yoshida,
Yuko Kawasaki,
Hironori Kobayashi,
Masatoshi Yamasaki,
Satoshi Nakayama,
Erika Miki,
Kei-Ichiro Shohmi,
Tomoko Matsushima,
Sachiyo Tada,
Yasuhiro Torikoshi,
Masakatsu Morita,
Shigeyuki Tamura,
Yoko Hino,
Jun Kamiyama,
Yoshihiro Sowa,
Yasunari Tsuchihashi,
Hisakazu Yamagishi,
Toshiyuki Sakai
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ABSTRACT: A series of molecular pathological investigations of the molecules that stimulate the cyclin dependent kinases (CDK1, 2, 4, and 6) have led to enormous accumulation of knowledge of the clinical significance of these molecules for cancer diagnosis. However, the molecules have yet to be applied to clinical cancer diagnosis, as there is no available technology for application of the knowledge in a clinical setting. We hypothesized that the direct measurement of CDK activities and expressions (CDK profiling) might produce clinically relevant values for the diagnosis. This study investigated the clinical relevance of CDK profiling in gastrointestinal carcinoma tissues by using originally developed expression and activity analysis methods. We have established novel methods and an apparatus for analyzing the expression and activities of the CDK molecules in lysate of tumor tissue in a clinical setting, and examined 30 surgically dissected gastrointestinal carcinomas and corresponding normal mucosal specimens. We demonstrate here that remarkably elevated CDK2 activity is evident in more than 70% of carcinoma tissues. Moreover, a G1-CDK activity profiling accurately mirrored the differences in proliferation between tumor and normal colonic tissues. Our results suggest that CDK profiling is a potent molecular-clinical approach to complement the conventional pathological diagnosis, and to further assist in the individualized medications.
Biochimica et Biophysica Acta 10/2005; 1741(3):226-33. · 4.66 Impact Factor
