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- Materials and Methods
Cyclin B1 is translocated to the nucleus from the cytoplasm, and plays an essential role in cell proliferation through promotion of mitosis. Although overexpression of cyclin B1 was previously reported in breast carcinomas, the biological significance of the intracellular localization of cyclin B1 remains unclear. Therefore, in this study, we examined cyclin B1 immunoreactivity in 109 breast carcinomas, according to the intracellular localization, that is, nucleus, cytoplasm or total (nucleus or cytoplasm). Total cyclin B1 was detected in carcinoma cells in 42% of breast carcinomas examined, whereas nuclear and cytoplasmic cyclin B1 were positive in 17 and 35% of the cases, respectively. Total or cytoplasmic cyclin B1 were positively associated with histological grade, mitosis, Ki-67, p53, c-myc or 14-3-3σ, and inversely correlated with estrogen or progesterone receptor. Nuclear cyclin B1 was significantly associated with tumor size, lymph node metastasis, histological grade, mitosis, Ki-67 or polo-like kinase 1. Only nuclear cyclin B1 was significantly associated with adverse clinical outcome of the patients, and multivariate analyses of disease-free and overall survival demonstrated nuclear cyclin B1 as the independent marker. A similar tendency was detected in the patients receiving adjuvant therapy after surgery. These results suggest that an onocogenic role of overexpressed cyclin B1 is mainly mediated in nuclei of breast carcinoma cells, and the nuclear translocation is regulated by polo-like kinase 1 and 14-3-3σ. Nuclear cyclin B1-positive breast carcinoma is resistant to adjuvant therapy, and nuclear cyclin B1 immunoreactivity is a potent prognostic factor in breast carcinoma patients. (Cancer Sci 2007; 98: 644–651)
Breast cancer is one of the most common malignancies in women worldwide. Invasive breast cancer has been generally regarded as a disease that metastasizes in an early phase, and clinical outcome of breast carcinoma patients is markedly influenced not only by metastasis of the tumor but also by proliferation activity of the tumor.(1) In fact, a multitude of prognostic factors identified for breast cancer have been demonstrated to be directly or indirectly related to proliferation of breast carcinoma cells.
It is well-known that proliferation of carcinoma cells is closely associated with altered regulation of the cell cycle.(2) Cell cycle progression is mediated by activation of a highly conserved family of cyclin-dependent kinases (Cdk),(3) and activation of a Cdk requires binding to a specific regulatory subunit, named a cyclin. Among the cyclins, cyclin B1 plays an essential role as a mitotic cyclin in the entry of mitosis from G2 phase.(4) Overexpression of cyclin B1 has been reported in various human tumors, and some of these studies demonstrated the clinical significance of cyclin B1 as a poor prognostic factor for some cancers,(5–7) including lymph node-negative breast carcinoma.(8)
Cyclin B1 is initially localized in the cytoplasm, and is translocated to the nucleus at the beginning of mitosis.(9) Nuclear translocation of cyclin B1 is considered very important to facilitate access of the cyclin B–Cdc2 (also named Cdk1) complex to its nuclear substrate and promote mitosis.(4) Therefore, it becomes very important to examine the intracellular localization of cyclin B1 in tumor tissues, in order to obtain a better understanding of the biological roles of cyclin B1.(10) Previously, Winters et al. reported that nuclear cyclin B1 immunoreactivity was significantly associated with reduced disease-free survival of breast carcinoma patients in a log-rank analysis.(11) However, no other information is available regarding the intracellular localization of cyclin B1 in breast carcinoma tissue, and the biological significance of cyclin B1 remains unclear at this juncture. Therefore, in the present study, we examined the intracellular immunolocalization of cyclin B1, and correlated these findings with various clinicopathological parameters of the patients, including their clinical outcome.
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- Materials and Methods
In the present study, cyclin B1 immunoreactivity was significantly associated with histological grade, mitotic count and Ki-67 LI in all intracellular components (i.e. total, nucleus and cytoplasm) of the breast carcinoma cases examined. Antibody Ki-67 recognizes cells in all phases of the cell cycle except G0 (resting) phase,(20) and Ki-67 LI is closely correlated with the S phase fraction and mitotic index.(1) Previously, Dutta et al. reported a positive correlation between cyclin B1 immunoreactivity and Ki-67 in breast carcinomas,(21) and Kuhling et al. showed that total cyclin B1 immunoreactivity is significantly associated with Ki-67 LI and histological grade in lymph node-negative breast carcinomas.(22) The results of our present study are in good agreement with these previous studies. Total cyclin B1 immunoreactivity is considered to reflect the physiological amount or aberrant expression of cyclin B1 protein,(22) and therefore, overexpression of cyclin B1 is postulated to play an important role in increased cell proliferation activity of human breast carcinoma.
The results of our study also demonstrated a significant association between total cyclin B1 and p53 or c-myc. Previous in vitro studies demonstrated that expression of cyclin B1 is suppressed by wild-type p53,(14,15,23) but is induced by mutant p53 or inactivation of p53.(24) The p53 antibody used in the present study (DO7) recognizes both the wild-type and mutated p53 proteins, but the accumulation of p53 protein is considered to be a good indicator of p53 mutation in breast carcinoma.(25) In addition, the cyclin B1 gene is a direct transcriptional target of c-myc,(24) and overexpression of c-myc has been reported to induce cyclin B1 expression.(16) The results of our present study as well as the in vitro studies above all indicate that overexpression of cyclin B1 is, at least in part, regulated by mutant p53 and c-myc proteins in breast carcinoma.
In our present study, nuclear cyclin B1 was significantly associated with tumor size, lymph node metastasis and adverse prognosis, but total or cytoplasmic cyclin B1 was not associated with these clinicopathological factors. Regarding the relationship between intracellular localization of cyclin B1 and the clinical outcome of breast carcinoma, Winters et al. reported that both nuclear and cytoplasmic cyclin B1 were associated with reduced disease-free or overall survival in their univariate analyses, but a significant association was only detected between nuclear cyclin B1 and disease-free survival in log-rank analyses.(11) These findings were partly consistent with the results of our present study. Cytoplasmic cyclin B1 may induce mitosis, but it is much weaker than nuclear cyclin B1.(15) In addition, Nozoe et al.(10) reported that the prognosis in esophageal carcinomas with nuclear-dominant expression of cyclin B1 is significantly worse than that of tumors with cytoplasmic-dominant expression. Therefore, the malignant potential of cyclin B1 may be mainly mediated by nuclear cyclin B1 in breast carcinoma cells, and cyclin B1 immunoreactivity is required to be evaluated in the nucleus, rather than total or cytoplasm, in breast carcinoma.
The mean value of nuclear cyclin B1 LI was only approximately half that of total or cytoplasmic cyclin B1 LI in our study, which suggests that the biological functions of overexpressed cyclin B1 may be regulated by nuclear transportation from the cytoplasm. Previous in vitro studies demonstrated that nuclear entry of cyclin B1 was facilitated by PLK1 through the phosphorylation of cyclin B1,(17,18) and overexpression of PLK1 was also reported in breast carcinoma.(26,27) However, 14-3-3σ anchored cyclin B1 in the cytoplasm and prevented the nuclear transition of cyclin B1 or inhibited mitosis.(19,28) In our present study, a significant association was detected between nuclear cyclin B1 and PLK1, and between cytoplasmic cyclin B1 and 14-3-3σ immunoreactivity. These results are consistent with previous in vitro studies, and PLK1 and 14-3-3σ may play important roles in the regulation of intracellular localization of cyclin B1 in human breast carcinoma cells.
The results of our univariate analyses revealed that the prognostic value of nuclear cyclin B1 was more significant than that of other proliferation markers, such as mitotic count and Ki-67. Nuclear cyclin B1 was significantly associated with adverse clinical outcome of the patients showing high (more than 5 cells) mitotic count in breast carcinoma, and multivariate analyses demonstrated that nuclear cyclin B1 was an independent poor prognostic factor in both recurrence and overall survival of the patients as well as lymph node metastasis, a well-established diagnostic modality.(29) This may be partly due to the fact that nuclear cyclin B1 demonstrated worse prognosis even in a group of patients who received adjuvant therapy following surgery. Radiation or most anticancer drugs usually result in DNA strand breaks and induce cell cycle arrest or cell death. DNA damage of carcinoma cells by radiotherapy or chemotherapy resulted in the p53-mediated inhibition of cell cycle progression in either G1 or G2–M.(30,31) Irradiation of tumor cells was usually associated with a G2 delay, a cellular response to DNA damage that allows time for repair and prevents mitosis of damaged cells. However, overexpression of cyclin B1 did not eliminate this G2 delay in irradiated cells,(32) overrode G2-M arrest, and made the cells enter into mitosis regardless of the status of p53 expression.(33) Cyclin B1 depletion has also been reported to inhibit proliferation and induce apoptosis of human breast carcinoma cells.(34) Hassan et al. reported that head and neck squamous cell carcinoma tumors overexpressing cyclin B1 were resistant to radiotherapy, which is similar to the results of our present study.(35) Therefore, residual carcinoma cells following surgical treatment in nuclear cyclin B1-positive breast carcinomas may grow rapidly regardless of the adjuvant therapy, thereby resulting in an increased recurrence and poor prognosis of these patients.
Escape from G2–M arrest by overexpressed cyclin B1 may allow insufficient time for DNA repair and cause the accumulation of mutations. Previous in vitro studies demonstrated that elevated levels of cyclin B1 often precede the onset of tumor cell immortalization and aneuploidy,(24,36,37) and Kuhling et al.(22) reported that cyclin B1 immunoreactivity was significantly associated with DNA aneuploidy in lymph node-negative breast carcinomas. Therefore, nuclear cyclin B1 may induce chromosomal instability and enhance the aggressiveness of the carcinoma cells. Further examination is required to clarify the detailed functions of nuclear cyclin B1 in breast carcinoma, in addition to its effects on cell proliferation.
In summary, nuclear cyclin B1 immunoreactivity was detected in carcinoma cells in 17% of human breast carcinomas, whereas total and cytoplasmic cyclin B1 immunoreactivities were detected in 42 and 35% of the cases, respectively. Cyclin B1 immunoreactivity in these three components (i.e. total, nucleus and cytoplasm) were all associated with histological grade, mitotic count or Ki-67 LI, and nuclear cyclin B1 was also correlated with tumor size and lymph node metastasis. Moreover, only nuclear cyclin B1 was significantly associated with adverse clinical outcome of the patients, and turned out to be an independent prognostic factor of both disease-free and overall survival by multivariate analyses. These results suggest that an onocogenic role of overexpressed cyclin B1 is mainly mediated in the nucleus of breast carcinoma cells, and nuclear cyclin B1 immunoreactivity is a potent prognostic factor in breast carcinoma patients.