Cyclin-dependent kinase 2 (cdk2) in the murine cdc2 kinaseTS mutant

Kinases of the mammalian cdc2 family including cdk2 (cyclin-dependent kinase 2) are thought to be involved in both the G₂/M transition and DNA replication. To investigate the role of cdc2 kinase and cdk2 in cell cycle progression, murine tsFT210 cells bearing a temperaturesensitive cdc2 mutation were used. These kinases were purified by column chromatography, using a peptide with the consensus phosphorylation site of cdc2 kinase as the substrate. In this mutant, cdc2 kinase activity was temperature sensitive and cdk2 activity was not. At the restrictive temperature, the mutant was only arrested in the G₂ phase and not in the G₂-S phase, suggesting that cdk2 did not compensate for cdc2 kinase at the G₂/M transition but did function at the G₁-S phase. This suggestion was supported by the finding that transfection of cdk2 cDNA did not improve the growth of the mutant cell line at the restrictive temperature, although transfection of cdc2 cDNA did.     

Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence

Endothelial cells (ECs) damage is an initial and pivotal step in the formation of atherosclerosis. Endothelial progenitor cells (EPCs), which have been considered as the precursor of ECs, can migrate and home to the site of injured ECs to divide into mature ECs and keep the integrity of the endothelial monolayer. It has been shown that the number and function of EPCs are negatively correlated with various atherosclerotic risk factors. This finding may be explained partly by accelerated senescence of EPCs induced by telomere attrition or shortening owning to oxidative stress and accumulative ROS. However, elevated telomerase activity which extends the telomere cannot lead to cellular immortal in the presence of the cyclin-dependent kinase inhibitor p16(INK4a). Researchers have the opinion that senescence is the balance between the regeneration and cancer. High expression of phosphorylated p16(INK4a), which is caused by oxidative stress and accumulative ROS, can prevent tumor cells from unlimited division and becoming malignant ones by accelerating premalignant cells premature senescence. It has been demonstrated that the expression of p16(INK4a) increases remarkably with age due to oxidative stress and accumulative ROS in some stem and progenitor cells, and regulates these cells age-dependent senescence. It is observed that telomeres shortening exists in these cells. Therefore, it can be hypothesized that p16(INK4a), together with telomerase, may co-modulate EPCs senescence.     

Amylin in pancreatic islets and pancreatic endocrine neoplasms

Amylin is a chief constituent of the amyloid present in insulinomas, and is colocalized in beta islet cells. By immunocytochemical staining, all four islet cells including insulin, glucagon, somatostatin (SRIF) and pancreatic polypeptide (PP) cells were positively stained for amylin. The strongly insulin-positive cells corresponded with the strongly amylin-positive cells, and glucagon cells appeared to be strongly positive for amylin, whereas SRIF and PP cells were weakly positive for amylin. Among 37 cases of pancreatic endocrine neoplasms, insulinomas were more stronger stained for amylin than other islet cell tumors; however, amylin staining was the same or weaker than insulin staining. Glucagonomas and PP-omas were weakly positive for amylin, whereas six of 11 gastrinomas were weakly positive for amylin. It is concluded that three orthoendocrine tumors including insulinomas, glucagonomas and PP-omas were all positive for amylin, whereas ectopic hormone secreting gastrinomas were positive for amylin in six of 11 cases (55%). This colocalization of amylin with insulin, glucagon and PP may support a structure-function relationship of amylin and pancreatic hormones. The lesser immunoreactive amylin in pancreatic endocrine neoplasms than in normal islet cells may contribute to autonomous hypersecretion of hormones by pancreatic endocrine neoplasms.     

Metallothionein in pancreatic endocrine neoplasms.

Metallothioneins (MTs) are intracellular proteins that bind to metal ions and are involved in heavy metal homeostasis and detoxification. Pancreatic islets were shown to be positive for zinc-containing matrix metalloproteinase-2 and -9 by immunocytochemical staining. The immunolocalization of matrix metalloproteinases in pancreatic islets prompted us to study further the link between zinc and MT in 34 cases of pancreatic endocrine neoplasms, including insulinomas, glucagonomas, gastrinomas, pancreatic polypeptide-omas, and non-functioning endocrine neoplasms. Four types of islet cells were found to be positive for MT, whereas pancreatic endocrine neoplasms mostly were either weakly positive or negative for MT. The presence of MT in normal islet cells and pancreatic endocrine neoplasms is consistent with the notion that MTs modulate zinc homeostasis and metabolism in pancreatic islet cells and pancreatic endocrine neoplasms as those tissues contain zinc-containing matrix metalloproteinases.     

Serotonin immunoreactivity in pancreatic endocrine neoplasms (carcinoid tumors).

Primary serotonin secreting pancreatic endocrine neoplasms (carcinoid tumors) are extremely rare and may be associated with manifestations of the carcinoid syndrome. Two cases of primary carcinoid tumor of the pancreas with liver metastases showed clinical and biochemical features of the carcinoid syndrome. Both cases demonstrated strong positive immunoreactivity for serotonin within the tumor cells. In an attempt to determine the relationship between pancreatic carcinoid tumors and other pancreatic endocrine neoplasms, immunostains for serotonin were performed on 11 additional islet cell tumors and on non-neoplastic pancreatic tissues. These cases showed serotonin immunoreactivity within islet cell tumors (36%). In addition, focal staining for serotonin was present in non-neoplastic ducts and ductules (88%), acini (22%), and islets of Langerhans (33%). Based on these observations, specific criteria are suggested for the diagnosis of primary pancreatic carcinoid tumor.     

细胞周期素依赖性激酶2及p27Kipl与血管瘤

目的:进一步探讨细胞周期素依赖性激酶2(CDK2)及p27Kipl.在血管瘤发生、发展及退化过程中的作用机制.方法:采用免疫组织化学SP法检测49例皮肤毛细血管瘤增生期、退化期及正常皮肤组织中CDK2和p27Kipl的表达水平;采用HPIAS-1000高清晰度彩色病理图文报告管理系统,对CDK2和p27Kipl表达的平均光密度和阳性面积率进行图像分析.结果:增生期组CDK2的表达明显高于退化期组和正常皮肤组织组;增生期血管瘤内皮细胞p27Kipl的表达显著低于退化期血管瘤内皮细胞.结论:p27Kipl能抑制血管瘤内皮细胞的增殖,在血管瘤的退化过程中起了重要作用;而CDK2能促进血管瘤内皮细胞增殖,存血管瘤的增生过程巾起了重要作用.     

Loss of p27 nuclear expression in a prognostically favorable subset of well-differentiated pancreatic endocrine neoplasms

Decreased expression of p27 occurs in aggressive colon, breast, and prostate neoplasms; p27 loss often correlates with worsened prognosis. Paradoxical overexpression has been described in benign and malignant pancreatic endocrine neoplasms (PENs). To investigate prognostic usefulness of p27 expression in PENs, we immunolabeled 42 primary PENs, with or without metastases, for p27 and separated lesions using a nuclear labeling index (NLI) of 10%. Of the 42 lesions, 26 demonstrated a 10% or higher NLI and 16 an NLI less than 10%. Comparison of lymph node status revealed that 50% of primary PENs with a 10% or higher NLI (13/26) demonstrated lymph node metastases, whereas only 6% of lesions with an NLI of less than 10% (1/16) demonstrated lymph node metastases (P = .0067). We next examined 11 liver and 7 lymph node metastases for p27 immunolabeling to determine whether p27 also is paradoxically retained in lesions that have metastasized. All 18 lesions demonstrated an NLI of 10% or higher for p27. Expression of p27 protein therefore appears to be lost in a subset of well-differentiated PENs with indolent features but paradoxically retained in PENs associated with metastatic disease.     

Chromosome 3p alterations in pancreatic endocrine neoplasia

Pancreatic endocrine tumors (PET) are rare neoplasms classified as functioning (F-PET) or non-functioning (NF-PET) according to the presence of a clinical syndrome due to hormonal hypersecretion. PETs show variable degrees of clinical aggressiveness and loss of chromosome 3p has been suggested to be associated with an advanced stage of disease. We assessed chromosome 3p copy number in 113 primary PETs and 32 metastases by fluorescence in situ hybridization (FISH) using tissue microarrays. The series included 56 well-differentiated endocrine tumors (WDET), 62 well-differentiated endocrine carcinomas (WDEC), and 6 poorly differentiated endocrine carcinomas (PDEC). Chromosome 3p alterations were found in 23/113 (20%) primary tumors, with losses being predominant over gains (14% vs. 6%). Loss of 3p was found in 5/55 (9%) WDET, 11/52 (21%) WDEC, and never in PDEC. Gains of 3p were detected in 4/55 (7%) WDET, no WDEC, but notably in 3/6 (50%) PDEC (OR 23.6; P = 0.003). Metastases were more frequently monosomic for 3p compared to primary tumors (OR 3.6; P = 0.005). Monosomy was significantly associated with larger tumor size, more advanced tumor stage, and metastasis. No association was found with survival. Chromosome 3p copy number alterations are frequent events in advanced stage PET, with gains prevailing in PDEC while losses are more frequent in WDEC, supporting the view that a specific pattern of alterations are involved in these diverse disease subtypes.     

The t(6;9)(p22;q34) in myeloid neoplasms: a retrospective study of 16 cases

Among patients with acute myeloid leukemia (AML), the t(6;9) (p22;q34) is a rare but defined subset with a poor prognosis. We report 16 patients with the t(6;9), of whom 13 had AML, 2 had myelodysplastic syndrome (MDS), and 1 had chronic myeloid leukemia in myeloid blast crisis (CML-BC). All except for one were evaluated at diagnosis. The median age was 34.5 (range: 7–62 years), with 12 adults and 12 males. Trilineage dysplasia was present in 13 (81%). Marrow basophilia was seen in only two patients, one of whom had CML-BC. HLA-DR was positive in all 12 patients assessed, CD33 in 11, CD13 in 10, and CD34 in seven. Four patients had one other abnormality apart from the t(6;9). These were the t(9;22) in the patient with CML and deletion 9q, addition 13q, and an isochromosome 8q in the other three patients. There were no complex karyotypes. Fms-related tyrosine kinase 3—internal tandem duplication (FLT3-ITD) mutations were seen in seven of 13 patients. Follow-up details were available for six patients. Three received palliative care, and follow-up details were not available for the other seven. The response to chemotherapy was poor in the remaining patients. The only patients who survived were three out of the four who had allogeneic hematopoietic stem cell transplantation (HSCT).     

Cyclin-dependent kinase inhibitor p27(Kip1) expression in thyroid cells obtained by fine-needle aspiration biopsy: a preliminary report

Recent studies on paraffin-embedded tissue have shown that the cyclin-dependent kinase inhibitor p27(Kip1) is expressed in normal thyroid cells, whereas it is downregulated in neoplastic cells. This prospective study was undertaken to assess whether p27(Kip1) staining may also be applied to fine-needle aspiration biopsy (FNAB) samples of the thyroid. We present here our preliminary results on 100 FNABs examined for p27(Kip1) expression. p27(Kip1) expression was assessed by immunocytochemistry; the technique was optimized on smears prepared from a normal thyreocyte cell line (TL5), which conspicuously expresses p27(Kip1), and then applied to FNAB samples prospectively collected from 80 cases of nodular goiter and 20 cases of thyroid neoplasms (10 papillary carcinomas and 10 follicular neoplasms). The TL5 cell culture smears showed that methanol fixation, followed by heat-induced antigen retrieval, is the most suitable technique for p27(Kip1) staining on cytological samples. The FNAB smears similarly treated showed high p27(Kip1) expression (75%) in goiter and a significantly lower expression (35%) in neoplasms (P < 0.0001). Our preliminary results show that: 1) p27(Kip1) protein expression can be reliably assessed on cytological samples; and 2) p27(Kip1) stains nonneoplastic and neoplastic samples in a different fashion, and thus is a useful tool in thyroid cytology.     

Human papilloma virus (HPV) status, p16INK4a, and p53 overexpression in epithelial malignant and borderline ovarian neoplasms

This investigation is the first to evaluate simultaneously human papilloma virus (HPV) status, p16(INK4a), and p53 immunoreactivity in epithelial ovarian neoplasms. The results were analyzed and correlated with histological type, histological grade, and survival of patients. Subtypes considered are papillary serous and mucinous. Polymerase chain reaction (PCR) analysis, performed in our previous study, had already demonstrated a small number of HPV-positive epithelial ovarian neoplasms. No significant correlation was found between the presence of HPV DNA and subtypes of ovarian neoplasms; thus, HPV cannot be considered responsible for epithelial ovarian neoplasm. Since p16 immunoreactivity was present in many other HPV-negative cases of epithelial ovarian neoplasms, this study suggests that p16 overexpression in some neoplasms of the female genital tract is not related to HPV carcinogenesis. A higher p53 expression rate observed between borderline and malignant serous tumors and between serous and mucinous neoplasms can confirm a recent dualistic model of ovarian carcinogenesis. According to this theory, low-grade serous carcinomas (serous intraepithelial carcinomas, serous borderline neoplasm, and ovarian mucinous neoplasms) (type I tumors) develop from mutations of KAS and BRAF, while high-grade serous carcinomas (type II tumors) develop from mutation of p53. In malignant neoplasms, for univariate analysis, patient survival seems to be related to p53, strong and diffuse p16 overexpression, and the stage of development of neoplasms at the diagnosis. In multinomial logistic regression, used to evaluate the role of staging, grading, p16 and p53 immunopositivity as predictor variables of unfavorable outcome of the disease, only p16 positivity was significantly related to the poor prognosis of the cancer.     

细胞周期蛋白依赖性蛋白激酶4抑制因子a与肿瘤

INK4a/ARF基因位点细胞特殊,它至少编码P16^INK4a和P19^ARF两种在调控细胞周期和细胞凋亡中起重要作用的蛋白质,人类的许多肿瘤与该位点的基因突变有关,现就这一基因位点的结构、功能和抑癌机理进行综述。     

Cyclin-dependent kinase inhibitor p27Kipl expression and interaction with other cell cycle-associated proteins in mammary carcinoma

p27, cyclin D1, and retinoblastoma (Rb) protein have been demonstrated using immunohistochemistry in 189 cases of primary breast carcinoma with long-term follow-up. There was a statistically significant association between the expression of p27 and both cyclin D1 and the retinoblastoma gene product (pRb), corresponding to their close interactions in regulating the G₁/S transition in the cell cycle. Low levels of p27 were seen in high-grade, rapidly proliferating, oestrogen receptor-negative tumours. In univariate analysis, low p27 expression was associated with a reduced relapse-free and overall survival. In multivariate analysis, p27 was not an independent predictor of survival when either histological grade or proliferative activity (S-phase fraction) was included in the model. When the combined expression of p27 and cyclin D1 was related to survival, patients with high levels of p27, regardless of their cyclin D1 status, did well, whilst those with low p27 had a poor outcome. The only exception, in the latter group, was patients with tumours expressing high levels of cyclin D1, who did as well as the high p27 group. We have shown that in clinical material p27 expression is associated with proliferative activity and while univariate analysis shows it to be a significant indicator of prognosis, this significance is lost in multivariate analysis when traditional prognostic factors are included in the model. The interest in p27 expression in mammary carcinoma lies in its behaviour when examined in combination with other G₁ cell cycle regulators. Copyright © 1999 John Wiley & Sons, Ltd.     

Study on expression of p16 and human papillomavirus 16 and 18 (E6) in OLP and its malignant transformation

Objective

The aim of this study is to investigate the expressions of p16 and HPV16/18(E6) in oral lichen planus (OLP) and malignant transformed OLP (MT-OLP).

血清饥饿条件下p21活化激酶6(PAK6)与雄激素受体表达的相关性及其意义

目的血清饥饿条件诱导前列腺癌细胞系中内源性p21活化激酶6(PAK6)与雄激素受体(AR)蛋白表达,探讨PAK6与AR蛋白表达的关系。方法在前列腺癌细胞系CWR22Rv1和LNcap细胞进行血清饥饿,诱导内源性PAK6与AR蛋白表达发生变化。结果血清饥饿处理的不同时间点,CWR22Rv1和LNCaP细胞中PAK6蛋白表达呈上升趋势,AR蛋白表达呈下降趋势。瞬时转染递增剂量的PAK6引起AR蛋白表达的抑制。结论血清饥饿条件诱导前列腺癌细胞中PAK6与AR蛋白表达呈负相关。1     

Cyclin-dependent kinase inhibitor p27Kip1 controls growth and cell cycle progression in human uterine leiomyoma

The molecular mechanism of the cell-cycle machinery in uterine leiomyoma has not yet been fully elucidated. Among the various types of cell-cycle regulators, p27(Kip1) (p27) is considered to be a potent tumor suppressor. To provide further molecular basis for understanding the progression of uterine leiomyoma, our objective was to evaluate the expression level of p27 in normal myometrium and uterine leiomyoma tissue and its effect on cytogenic growth. Western blot analysis, real-time polymerase chain reaction (PCR) and immunohistochemical staining revealed that p27 protein and messenger RNA were down-regulated in uterine leiomyoma tissue and cultured cells compared to normal myometrium. Full-length human p27 cDNA was transferred using a replication-deficient recombinant adenoviral vector (Ad.p27) into uterine leiomyoma cells and evaluated the effect on cell proliferation. Transfection of Ad.p27 into uterine leiomyoma cells resulted in the induction of apoptosis, reduction in viability and proliferation of uterine leiomyoma cells. Our results suggest a new paradigm that down-regulated p27 protein expression is the possible underlying mechanism for the growth of uterine leiomyoma and over-expression of p27 induces cell death. This study provides better understanding of the control exerted by p27 in regulating growth and disease progression of uterine leiomyoma.     

Cyclin-dependent kinase 5 activator p35 over-expression and amyloid beta synergism increase apoptosis in cultured neuronal cells

Alzheimer disease (AD) is a neurodegenerative disorder characterized by neuronal loss, dementia and pain. Two main protein aggregates, extracellular (senile plaques, SP) and intracellular (neurofibrillary tangles, NFT), are associated with AD. NFT are mainly composed of hyperphosphorylated microtubule-associated protein tau. Nowadays several protein kinases have been implicated in the phosphorylation of tau, including glycogen synthase kinase 3 beta (GSK3β), MAP kinase, protein kinase A and cyclin-dependent kinase 5 (Cdk5). A deregulation in the activity of Cdk5 has been postulated to participate in the abnormal tau hyperphosphorylation in AD. Activation of Cdk5 occurs after its association with p35, a neuron-specific activator, predominantly in the nervous system. Therefore, in this study we used the tetracycline transactivator system to increase p35/GFP in neuronal cells, treated with amyloid beta 1-42 (Aβ_1-42) peptide. These cells showed an increase of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase-3 staining, indicating increased apoptosis of neuronal cells. This effect could be reversed by the addition of tetracycline in the culture medium, suggesting synergistic effects of p35 over-expression and Aβ treatment in the apoptosis of neuronal cells. These results represent a linkage between amyloidogenic and cdk5 pathways leading to apoptosis of neuronal cells.     

Liver metastases arising from well-differentiated pancreatic endocrine neoplasms demonstrate increased VEGF-C expression.

Pancreatic endocrine neoplasms (PENs) are uncommon, generally well-differentiated neoplasms that demonstrate prominent endocrine differentiation. Although the majority of PENs remain localized, malignant spread may occur via lymphatic or hematogenous routes. Angiogenic growth factors, including the vascular endothelial growth factor (VEGF) family, have been implicated in new vessel growth and hematogenous metastases, although this has not been studied in PENs. We therefore examined 19 primary well-differentiated PENs and 7 liver metastases to determine the expression of VEGF-A and its family member VEGF-C by immunolabeling analysis. VEGF-A immunoreactivity was evident only in scattered cells throughout all lesions. VEGF-C, however, demonstrated low-to-moderate expression in primary PENs by semiquantitative histoscore analysis (factor of labeling intensity by percentage of positive cells), with significantly increased expression in liver metastases (mean histoscore indices: primary PEN, 4.7 versus liver metastases, 9.5; Student's t test; P =.002773). Microvascular density of primary PENs and liver metastases did not appear to linearly correlate with VEGF-C expression. Examination of the VEGF-C-specific receptors VEGFR-2/KDR/Flk-1 and VEGFR-3/Flt-4 demonstrated intense endothelial immunoreactivity for VEGFR-2, as well as VEGFR-2 and -3 expression on the majority of neoplastic cells, suggesting a possible role in autocrine/paracrine neoplastic growth regulation. We postulate that the upregulation of VEGF-C may be involved in PEN progression and metastases, although not via a direct proangiogenic mechanism.     

46，XY，t（4；6）（p16；p12）一例

患者 男，34岁。结婚近4年，其妻自然流产4次，均于孕50～70天出现阴道流血，经B超检查证实只有空孕囊无胚胎发育而行清宫术。夫妻表型智力正常、身体健康。非近亲婚配，其妻自诉孕期无有害物质接触及感染史，患者精液分析及前列腺液涂片分析均正常。患者父母无近亲关系和不良生育史，兄妹3人所生子女表型及智力均无异常。     

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.