Expression Of CDC25B Research Articles

Growth hormone stimulates proliferation of old-aged regenerating liver through forkhead box m1b

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA-binding domain and the members of which play essential roles in cellular proliferation, differentiation, and longevity. Reduced cellular proliferation during aging is associated with a progressive decline in both growth hormone (GH) secretion and Foxm1b expression. Liver regeneration studies with 12-month-old (old-aged) transgenic mice indicated that increased hepatocyte expression of Foxm1b alone is sufficient to restore hepatocyte proliferation to levels found in 2-month-old (young) regenerating liver. GH therapy in older people has been shown to cause an increase in cellular proliferation, but the transcription factors that mediated this stimulation in proliferation remain uncharacterized. In this study, we showed that human GH administration to old-aged Balb/c mice dramatically increased both expression of Foxm1b and regenerating hepatocyte proliferation. This increase in old-aged regenerating hepatocyte proliferation was associated with elevated protein expression of Cdc25A, Cdc25B, and cyclin B1, with reduced protein levels of cyclin-dependent kinase inhibitor p27 Kip1 (p27). GH treatment also was found to stimulate hepatocyte proliferation and expression of Foxm1b protein without partial hepatectomy (PHx). Furthermore, GH treatment of young Foxm1b −/− mice failed to restore regenerating hepatocyte DNA replication and mitosis caused by Foxm1b deficiency. These genetic studies provided strong evidence that the presence of Foxm1b is essential for GH to stimulate regenerating hepatocyte proliferation. In conclusion, our old-aged liver regeneration studies show that increased Foxm1b levels are essential for GH to stimulate hepatocyte proliferation, thus providing a mechanism for GH action in the elderly. (H epatology 2003;38:1552-1562.)

Growth Hormone Stimulates Proliferation of Old–Aged Regenerating Liver Through Forkhead Box M1b

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA-binding domain and the members of which play essential roles in cellular proliferation, differentiation, and longevity. Reduced cellular proliferation during aging is associated with a progressive decline in both growth hormone (GH) secretion and Foxm1b expression. Liver regeneration studies with 12-month-old (old-aged) transgenic mice indicated that increased hepatocyte expression of Foxm1b alone is sufficient to restore hepatocyte proliferation to levels found in 2-month-old (young) regenerating liver. GH therapy in older people has been shown to cause an increase in cellular proliferation, but the transcription factors that mediated this stimulation in proliferation remain uncharacterized. In this study, we showed that human GH administration to old-aged Balb/c mice dramatically increased both expression of Foxm1b and regenerating hepatocyte proliferation. This increase in old-aged regenerating hepatocyte proliferation was associated with elevated protein expression of Cdc25A, Cdc25B, and cyclin B1, with reduced protein levels of cyclin-dependent kinase inhibitor p27(Kip1) (p27). GH treatment also was found to stimulate hepatocyte proliferation and expression of Foxm1b protein without partial hepatectomy (PHx). Furthermore, GH treatment of young Foxm1b -/- mice failed to restore regenerating hepatocyte DNA replication and mitosis caused by Foxm1b deficiency. These genetic studies provided strong evidence that the presence of Foxm1b is essential for GH to stimulate regenerating hepatocyte proliferation. In conclusion, our old-aged liver regeneration studies show that increased Foxm1b levels are essential for GH to stimulate hepatocyte proliferation, thus providing a mechanism for GH action in the elderly.

Alteration of gene expression during radiation-induced resistance and tumorigenesis in NIH3T3 cells revealed by cDNA microarrays: involvement of MDM2 and CDC25B.

To identify a set of genes involved in the development of radiation-induced tumorigenesis, we used DNA microarrays consisting of 1176 mouse genes and compared expression profiles of radioresistant cells, designated NIH3T3-R1 and NIH3T3-R4. These cells were tumorigenic in a nude mouse grafting system, as compared with the parental NIH3T3 cells. Expression of MDM2, CDK6 and CDC25B was found to increase more than 3-fold. Entactin protein levels were down-regulated in NIH3T3-R1 and NIH3T3-R4 cells. Changes in gene expression were confirmed by reverse transcription-PCR or western blotting. When these genes were transfected into NIH3T3 cells, CDC25B and MDM2 overexpressing NIH3T3 cells showed radioresistance, while CDK6 overexpressing cells did not. In the case of entactin, overexpressing NIH3T3-R1 and NIH3T3-R4 cells were still radioresistant. Furthermore, CDC25B and MDM2 overexpressing cells grafted into nude mice were tumorigenic. NIH3T3-R1 and NIH3T3-R4 cells showed increased radiation-induced apoptosis accompanied by a faster growth rate, rather than an earlier radiation-induced G2/M phase arrest, suggesting that the radioresistance of NIH3T3-R1 and NIH3T3-R4 cells was due to a faster growth rate rather than induction of apoptosis. In the case of MDM2 and CDC25B overexpressing cells, similar phenomena, such as increased apoptosis and a faster growth rate, were shown. The above results, therefore, demonstrate involvement of CDC25B and MDM2 overexpression in radiation-induced tumorigenesis and provide novel targets for detection of radiation-induced carcinogenesis.

Relationship between cyclin D1 and p21 Waf1/Cip1 during differentiation of human myeloid leukemia cell lines

Expression of cell cycle-regulating genes was studied in human myeloid leukemia cell lines ML-1, ML-2 and ML-3 during induction of differentiation in vitro. Myelomonocytic differentiation was induced by phorbol ester (12- o-Tetradecanoyl-phorbol-13-acetate, TPA), tumor necrosis factor α (TNFα) or interferon γ (INFγ), or their combination. Differentiation (with the exception of TNFα alone) was accompanied by inhibition of DNA synthesis and cell cycle arrest. Inhibition of proliferation was associated with a decrease in the expression of cdc25A and cdc25B, cdk6 and Ki-67 genes, and with increased p21 Waf1/Cip1 gene expression, as measured by comparative RT-PCR. Expression of the following genes was not changed after induction of differentiation: cyclin A1, cyclin D3, cyclin E1 and p27 Kip1. Surprisingly, cyclin D1 expression was upregulated after induction by TPA, TNFα with IFNγ or BA. Cyclin D2 was upregulated only after induction by BA. The results of the expression of the tested genes obtained by comparative RT-PCR were confirmed by quantitative real-time (RQ) RT-PCR and Western blotting. Quantitative RT-PCR showed as much as a 288-fold increase of cyclin D1 specific mRNA after a 24 h induction by TPA. The upregulation of cyclin D1 in differentiating cells seems to be compensated by the upregulation of p21 Waf1/Cip1. These results, besides others, point to a strong correlation between the expression of cyclin D1 and p21 Waf1/Cip1 on the one hand and differentiation on the other hand in human myeloid leukemic cells and reflect a rather complicated network regulating proliferation and differentiation of leukemic cells.

Prediction of the response to chemoradiation and prognosis in oesophageal squamous cancer.

The sensitivity of cancer cells to chemotherapy and radiation therapy depends on various biological properties. This study investigated the expression of p53, CDC25B and metallothionein (MT), and evaluated their clinical significance in chemoradiation therapy (CRT) for oesophageal squamous cell carcinoma. The expression of p53, CDC25B and MT was evaluated by immunohistochemistry using biopsy specimens taken before CRT for 77 patients with oesophageal squamous cell carcinoma, and correlated with the pathological effects of CRT and survival. p53-positive tumours and MT-positive tumours had a poor response to CRT, whereas tumours with strong CDC25B expression were associated with a good response. When each patient was scored for the presence of the three biological factors, there was a strong correlation between the sensitivity score and the pathological effect of CRT (P < 0.001), and a (non-significant) difference in the 5-year survival rate between patients with a high score and those with a low score (67 versus 34 per cent respectively; P = 0.12). The combined evaluation of p53, CDC25B and MT may help to identify patients with advanced oesophageal squamous cell carcinoma who will benefit from preoperative CRT.

Cdc25-dependent activation of cyclin A/cdk2 is blocked in G2 phase arrested cells independently of ATM/ATR.

Cyclin A/cdk2 is active during S and G2 phases of the cell cycle, but its regulation and function during G2 phase is poorly understood. In this study we have examined the regulation of cyclin A/cdk2 activity during normal G2 phase progression and in genotoxin-induced G2 arrest. We show that cyclin A/cdk2 is activated in early G2 phase by a cdc25 activity. In the G2 phase checkpoint arrest initiated in response to various forms of DNA damage, the cdc25-dependent activation of both cyclin A/cdk2 and cyclin B1/cdc2 is blocked. Ectopic expression of cdc25B, but not cdc25C, in G2 phase arrested cells efficiently activated both cyclin A/cdk2 and cyclin B1/cdc2. Finally, we demonstrate that the block in cyclin A/cdk2 activation in the G2 checkpoint arrest is independent of ATM/ATR. We speculate that the ATM/ATR-independent block in G2 phase cyclin A/cdk2 activation may act as a further layer of checkpoint control, and that blocking G2 phase cyclin A/cdk2 activation contributes to the G2 phase checkpoint arrest.

Increased susceptibility to carcinogen-induced mammary tumors in MMTV-Cdc25B transgenic mice.

Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) by dephosphorylating critical phospho-tyrosine and phospho-threonine residues on these proteins. Several types of studies indicate that Cdc25s can enhance cell proliferation and oncogenesis. Furthermore, overexpression of Cdc25A and/or B have been detected in several types of primary human cancers, including breast cancers. To further assess the oncogenic capacity of Cdc25B in vivo, we have generated transgenic mice that overexpress Cdc25B in the mammary epithelium, driven by the MMTV - LTR promoter. Although these mice are grossly normal for up to 18 months, the ectopic expression of Cdc25B in their mammary glands increases the susceptibility of these mice to induction of mammary tumors by the carcinogen 9,10-dimethyl-1, 2-benzanthracene (DMBA).

Induction of mammary gland hyperplasia in transgenic mice over-expressing human Cdc25B.

Cdc25 A and B are dual-specificity phosphatases which have been implicated in neoplastic transformation. Although Cdc25A and Cdc25B have been found to be over-expressed in many cancer cell lines and primary tumors, the physiological roles of Cdc25A and B in vivo are largely undefined. To investigate the roles of these proteins in the oncogenic transformation of the mammary gland we used the mouse mammary tumor virus (MMTV) promoter to target over-expression of the Cdc25B transgene in the mammary glands of transgenic mouse lines. Here we report that the over-expression of Cdc25B enhances the proliferation of mammary epithelial cells resulting in the formation of precocious alveolar hyperplasia. At the molecular level, marked increases in cyclin D1 protein have been found in transgenic mammary epithelial cells. The accelerated growth rate of the mammary epithelial cells could also be attributed to the increased levels of cyclin E/cdk2 activity. In addition, a pronounced decrease in apoptosis was also observed during the involution of mammary gland. The reduction of apoptosis during involution correlated well with the reduced expression of c-myc and p53, both of which have been implicated in apoptosis. Taken together, our results clearly indicate that the deregulated expression of Cdc25B generates mammary gland hyperplasia.

Overexpression of Cyclin‐dependent Kinase‐activating CDC25B Phosphatase in Human Gastric Carcinomas

CDC25 phosphatases activate cyclin‐dependent kinases by removing inhibitory phosphate groups on the molecules and positively regulate the cell cycle progression. The expression of CDC25A, B and C was examined in gastric carcinoma cell lines and gastric carcinoma tissues by northern blotting and immunohistochemistry. The gastric carcinoma cell lines expressed CDC25A, B and C mRNA at various levels. The expression levels of CDC25B were generally higher than those of CDC25A and C. Of the 40 gastric carcinomas, 70% of the tumors expressed CDC25B mRNA at higher levels than the corresponding normal mucosas, while 38% overexpressed CDC25A mRNA. The CDC25C expression was at very low or undetectable levels. No obvious correlation was detected between the expression of CDC25B and p53 gene mutations. Inununohistochemically, CDC25‐positive tumor cells were detected in 43 (78%) of 55 gastric carcinoma cases, of which 27 (49%) were strongly positive. Strong expression of CDC25B protein was associated with advanced stage and deep invasion. Furthermore, the incidence of strong expression was significantly higher in carcinomas with nodal metastasis than in those without metastasis. These findings suggest that Overexpression of CDC25B may favor development and progression and may be an indicator of malignant behavior of gastric carcinomas.

Cell cycle arrest in the G2 phase induced by phorbol ester and diacylglycerol in vascular endothelial cells.

The role of protein kinase C (PKC) in vascular endothelial cell proliferation was investigated using human umbilical vein endothelial cells released from the G1/S border. Phorbol 12-myristate 13-acetate (PMA) caused G2 arrest because 1) when added to G2 cells, PMA inhibited subsequent cell division; 2) these growth-arrested cells did not show morphological features of mitotic cells; and 3) PMA did not interrupt mitosis in cells released from nocodazole-induced M phase arrest. 1-Oleoyl-2-acetyl-sn-glycerol (OAG) added repeatedly from G2 also inhibited mitosis. The activation of cdc2 kinase around the G2/M transition was suppressed by PMA and OAG. Although cdc2 was expressed in the presence of PMA, dephosphorylation of its tyrosine residue was inhibited by PMA. In parallel, the expression of cdc25B was suppressed by PMA. The total and the cdc2-associated amount of cyclin B were both reduced by PMA. These data suggested that the PKC pathway negatively regulates the G2/M transition and that the inhibition of cdc2 kinase by the reduction in the levels of cdc25B and cyclin B may contribute to this effect.

The Distinct and Developmentally Regulated Patterns of Expression of Members of the Mouse Cdc25 Gene Family Suggest Differential Functions during Gametogenesis

Cdc25 genes encode protein threonine/tyrosine phosphatases that activate cyclin-dependent kinases. A cDNA encoding the murine homologue of the human Cdc25C gene was isolated in studies designed to identify those genes involved in regulating the mitotic and meiotic cell cycles of developing mouse germ cells. Northern blot and in situ hybridization along with immunoblot analyses were performed to determine the patterns of expression of Cdc25C compared to those of a second Cdc25 family member, Cdc25B . Murine Cdc25C transcripts of 2.1 kb were detected in midgestation embryos and in several adult tissues, including testis and ovary. The highest levels of Cdc25C transcripts were detected in the testis, with low levels of the 2.1-kb transcript and abundant levels of a 1.9-kb transcript that was not detected in the other tissues examined. In the testis, Cdc25C expression was localized in germ cells, specifically in late pachytene-diplotene spermatocytes and round spermatids, whereas Cdc25B expression was most readily detected in the somatic cells. In the ovary, Cdc25C expression was apparent in cumulus granulosa cells, whereas the expression of Cdc25B was detected in both growing oocytes and somatic cells, including the granulosa cells. These results not only indicate that the expression of the murine Cdc25 genes is lineage- and developmental stage-specific in the mouse testis and ovary but also suggest that the Cdc25 genes may have different functions in the germinal and somatic compartments of the ovary and the testis, only some of which are involved in cellular proliferation.