Immunohistochemical evaluation of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus

Control of the G1/S-phase transition as well as angiogenic switch are two of the most studied mechanisms in cancer. The current study examined the correlation between the immunohistochemical expression of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus (BE). Overall, p53 showed a much higher expression in BE patients (up to 50%) than in controls (1-10%) (P < 0.005). Also p21 showed a downregulation in BE when compared to normal esophagus (70% of cells vs. 65%), but the difference did not show any statistical significance (P = 0.45). pRb2/p130 was detected in 80% of cells in normal controls, but showed positive in only 20% of cells in BE biopsies. Additionally, Rb2/p130 expression was inversely correlated to that of VEGF, EZH2, and PCNA (P < 0.0001, P = 0.0032, P < 0.001, respectively). p27 stained more intensely and in a widespread manner (70%) cells in normal esophageal tissues but about only 30% in BE samples (P < 0.001). Lastly, in accordance with other reports, we also found p16 expressed by immunohistochemistry at high levels in normal controls and at low levels in BE (P < 0.001). In conclusion, p16, p21, p27, and p53 staining confirmed previously published data. Interestingly, pRb2/p130 expression was found significantly decreased in metaplastic epithelium compared to normal controls and showed significant inverse correlation with the expression of other markers, such as VEGF, EZH2, and PCNA. These data, taken together, indicate that these molecular events occurring in Barrett's metaplasia (BM) may represent one of the many steps taking place during esophageal malignant progression such as impairment of cell-cycle control, altered differentiation, and unbalanced angiogenesis.     

幽门螺杆菌感染与胃癌中p21和p27表达的关系

探讨胃癌中p21~(WAF1)和p27~(KIP1)的表达及其临床意义,并分析幽门螺杆菌(Hp)感染和p21~(WAF1)、p27~(KIP1)表达的关系。采用免疫组化法检测了89例胃癌组织中p21~(WAF1)和p27~(KIP1)表达水平,并采用快速尿素酶试验和组织病理学检测两种方法检查这些胃癌病例的Hp感染情况。实验结果显示,胃癌组织p21~(WAF1)的表达水平在不同病例组织中有所差异。结合临床病理学指数分析显示,降低的p21~(WAF1)表达与较深的肿瘤侵袭密切相关(P<0.05)。免疫组织化学结果显示,p27~(KIP1)无论在细胞浆中还是细胞核内都有表达,p27~(KIP1)核表达水平与胃癌的组织病理学分型密切相关(P<0.05)。此外,还发现Hp阳性病例的p27~(KIP1)阳性表达率明显低于Hp阴性者(P<0.05),而p21~(WAF1)表达阳性率在Hp阳性和阴性胃癌病例中无显著差异(P>0.05)。结果提示,胃癌的进展与细胞周期调节蛋白p21~(WAF1)和p27~(KIP1)的表达下调有关,Hp感染的致癌过程中可能有p27~(KIP1)参与。     

Critical role of both p27KIP1 and p21CIP1/WAF1 in the antiproliferative effect of ZD1839 ('Iressa'), an epidermal growth factor receptor tyrosine kinase inhibitor,in head and neck squamous carcinoma cells

High expression of the epidermal growth factor receptor (EGFR) has been implicated in the development of squamous-cell carcinomas of head and neck (SCCHN). ZD1839 ('Iressa') is an orally active, selective EGFR-TKI (EGFR-tyrosine kinase inhibitor) that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. We have demonstrated that ZD1839 induces growth arrest in SCCHN cell lines by inhibiting EGFR-mediated signaling. Cell cycle kinetic analysis demonstrated that ZD1839 induces a delay in cell cycle progression and a G1 arrest together with a partial G2/M block; this was associated with increased expression of both p27(KIP1) and p21(CIP1/WAF1) cyclin-dependent kinase (CDK) inhibitors. The activity of CDK2, the main target of CIP/KIP CDK inhibitors, was reduced in a dose-dependent fashion after 24 h of ZD1839 treatment and this effect correlated to the increased amount of p27(KIP1) and p21(CIP1/WAF1) proteins associated with CDK2-cyclin-E and CDK2-cyclin-A complexes. In addition, ZD1839-induced growth inhibition was significantly reduced in cell transfectants expressing p27(KIP1) or p21(CIP1/WAF1) antisense constructs. Overall, these results as well as the timing of the effect of ZD1839 on G1 arrest and p27(KIP1) and p21(CIP1/WAF1) upregulation, suggest a mechanistic connection between these events.     

Alterations in TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expression associated with progression in B-CLL

B-cell chronic lymphocytic leukaemia (B-CLL) originates from B lymphocytes that may differ in the activation level, maturation state or cellular subgroups in peripheral blood. Tumour progression in CLL B cells seems to result in gradual accumulation of the clone of resting B lymphocytes in the early phases (G0/G1) of the cell cycle. The G1 phase is impaired in B-CLL. We investigated the gene expression of five key cell cycle regulators: TP 53, c-Myc, cyclin D2, p21WAF1/CIP1 and p27KIP1, which primarily regulate the G1 phase of the cell cycle, or S-phase entry and ultimately control the proliferation and cell growth as well as their role in B-CLL progression. The study was conducted in peripheral blood CLL lymphocytes of 40 previously untreated patients. Statistical analysis of correlations of TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expressions in B-CLL patients with different Rai stages demonstrated that the progression of disease was accompanied by increases in p53, cyclin D2 and c-Myc mRNA expression. The expression of p27KIP1 was nearly statistically significant whereas that of p21 WAF1/CIP1 showed no such correlation. Moreover, high expression levels of TP53 and c-Myc genes were found to be closely associated with more aggressive forms of the disease requiring earlier therapy.     

Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.     

Osteoclast differentiation is associated with transient upregulation of cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1)

Osteoclasts, bone-resorbing multinucleated cells, develop from monocyte-macrophage lineage cells in the presence of osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) and macrophage colony-stimulating factor (M-CSF). M-CSF-dependent bone marrow macrophages (M-BMMPhis) from mouse bone marrow cells have been shown to differentiate into osteoclast-like multinucleated cells (OCLs) in the presence of soluble ODF/RANKL (sODF/RANKL) and M-CSF within 3 days. In this study, we found that stimulation of M-BMMPhis with sODF/RANKL induced a transient expression of cyclin-dependent kinase inhibitors (CDK inhibitors) p21(WAF1/CIP1) and p27(KIP1) by 24 h. The CDK inhibitor proteins disappeared by 48 h. Tumor necrosis factor alpha (TNF-alpha), which is reported to stimulate OCL differentiation, stimulated p21(WAF1/CIP1) and p27(KIP1) expression in M-BMMPhis as well. However, M-CSF alone did not stimulate the expression of the two CDK inhibitors. To clarify the role of p21(WAF1/CIP1) and p27(KIP1) in osteoclastogenesis, accumulation of these CDK inhibitors was aborted by antisense oligonucleotides. Treatment with p21(WAF1/CIP1) antisense oligonucleotide alone, or p27(KIP1) antisense oligonucleotide alone, showed a limited inhibitory effect on OCL formation. However, treatment with a mixture of these two antisense oligonucleotides strongly inhibited OCL formation. These results suggest that a combined modulation of the CDK inhibitors p21(WAF1/CIP1) and p27(KIP1) may be involved in osteoclast differentiation induced by ODF/RANKL.     

Antitumor protein (AP) from a mushroom induced apoptosis to transformed human keratinocyte by controlling the status of prb, c-MYC, cyclin E-cdk2, and p21WAF1 in the G1/S transition

Antitumor protein (AP) from a mushroom, induced the morphological changes typical to apoptosis such as nuclear condensation, aneuploidity, and DNA fragmentation at concentrations as low as 5-20 ng/ml to cancer cells. Molecular alterations related to cell cycle. Molecular alterations related to cell cycle, especially G1/S transition were investigated with a human keratinocyte transformed with oncoproteins, E6 and E7 of human pappiloma virus(HPV)-16. AP didn't alter significantly and oncosuppressor p53 level, but induced hyperphosphorylation of pRb. Time-dependent change of G1 cyclins, cdk2 and cdk4 after addition of AP showed that expression level of cdk inhibitors, INK4 family, and p27KIP1 did not altered, while that of p21WAF1 was downregulated.     

EZH2 Is Associated with Malignant Behavior in Pancreatic IPMN via p27Kip1 Downregulation

Background

The epigenetic mechanism of tumorigenesis in pancreatic intraductal papillary mucinous neoplasm (IPMN) remains largely unknown. The aim of this study is to examine the role of enhancer of zeste homologue 2 (EZH2) alteration in pancreatic IPMN progression.

Methods

Fifty-four surgically resected pancreatic IPMN specimens, including a total of 181 lesions (normal duct in 48, adenoma in 50, borderline atypia in 53, carcinoma in situ (CIS) in 19, and invasive carcinoma in 11) were analyzed by immunohistochemical staining (EZH2, Ki-67, p27^Kip1). Using paraffin embedded sections, total RNA was successfully extracted from 20 IPMN lesions (borderline IPMN in 9, CIS in 6, invasive carcinoma in 5) and 7 pancreatic normal ducts, and then levels of EZH2 and p27^Kip1 mRNA were analyzed by real time PCR.

Results

In immunohistochemical analysis, cell proliferative activity revealed by Ki-67 positive nuclei was increased during IPMN progression (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma). EZH2 expression displayed a similar pattern (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma) with cell proliferative activity. EZH2 expression in malignant (CIS and invasive carcinoma) IPMNs was significantly higher than that in adenoma and borderline-atypia IPMNs. EZH2 expression level in IPMN lesions was positively correlated with the Ki-67 positive nuclear ratio (p<0.0001). EZH2-positive cells in malignant IPMN did not express p27^Kip1. EZH2 mRNA expressions in malignant lesions were significantly higher than those in benign lesions (p<0.0001). In contrast, p27^Kip1 mRNA in malignant lesions was significantly decreased compared to those in benign lesion (p<0.05), and there was an inverse correlation between EZH2 and p27^Kip1 mRNA levels (p = 0.0109).

Conclusion

EZH2 is associated with the accelerated cell proliferation and malignant step in pancreatic IPMN via the downregulation of p27^Kip1.     

Activation of the anaphase promoting complex by HTLV-1 tax leads to senescence

The human T-lymphotropic virus type 1 (HTLV-1) Tax binds the anaphase promoting complex (APC) and activates it ahead of schedule. Here, we show that APC activation by Tax induces rapid senescence (tax-IRS) independently of p53 and pRB. In response to tax, cyclin A, cyclin B1, securin, and Skp2 becomes polyubiquitinated and degraded starting in S phase. This is followed by a surge in p21(CIP1/WAF1) and p27(KIP1) in mid to late S and G2/M leading to a permanent G1 arrest. Tax-positive HTLV-1-transformed T-cell lines express elevated levels of p21(CIP1/WAF1), but low levels of p27(KIP1). Finally, Tax can be stably expressed in p27(KIP1)-null NIH3T3 cells. These results indicate that APC activation by Tax causes inactivation of SCF(Skp2) and stabilization of p21(CIP1/WAF1) and p27(KIP1). The build-up of p21(CIP1/WAF1) and especially p27(KIP1) commits cells to senescence. Evading tax-IRS through a loss of p27(KIP1) function is likely to be critical for cell transformation by Tax and development of adult T-cell leukemia after HTLV-1 infection. Finally, activation of APC ahead of schedule may be exploited to arrest cancer cell growth.     

Retracted: Long noncoding RNA TALNEC2 plays an oncogenic role in breast cancer by binding to EZH2 to target p57KIP2 and involving in p-p38 MAPK and NF-κB pathways

We aimed to investigate the potential role and regulatory mechanism of long noncoding RNA tumor-associated lncRNA expressed in chromosome 2 (TALNEC2) in breast cancer. The expression of TALNEC2 in breast cancer tissues and cells were investigated. MCF-7 and MDA-MB-231 cells were transfected with small interfering RNA (siRNA) duplexes for targeting TALNEC2 (si-TALNEC2), enhancer of zeste homolog 2 (EZH2; si-EZH2) and p57^KIP2 (si-p57 ^KIP2), and their corresponding controls (si-NC). The viability, colony forming ability, cell cycle, apoptosis, and autophagy of transfected cells were assessed. The expressions of p-p38 mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathway-related proteins were investigated. The results showed that TALNEC2 was highly expressed in breast cancer tissues and cells. Knockdown of TALNEC2 significantly inhibited the malignant behaviors of MCF-7 and MDA-MB-231 cells, including inhibiting cell viability and colony forming, arresting cell cycle at G0/G1 phase, inducing cell apoptosis, and promoting cell autophagy. EZH2 was a TALNEC2 binding protein, which was upregulated in breast cancer tissues and cells and could negatively regulate p57 ^KIP2. Effects of TALNEC2 knockdown on malignant behaviors of MCF-7 cells were reversed by p57 ^KIP2 knockdown. The expressions of p-p38, RelA, and RelB in MCF-7 cells were decreased after knockdown of TALNEC2 or EZH2, which were reversed by knockdown of p57 ^KIP2 concurrently. In conclusion, TALNEC2 may play an oncogenic role in breast cancer by binding to EZH2 to target p57 ^KIP2. Activation of p-p38 MAPK and NF-κB pathways may be key mechanisms mediating the oncogenic role of TALNEC2 in breast cancer. TALNEC2 may serve as a promising target in the therapy of breast cancer.     

Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1). This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.     

Low levels of p27 in association with deregulated p53-pRb protein status enhance tumor proliferation and chromosomal instability in non-small cell lung carcinomas

BACKGROUND: Down-regulation or overexpression of the cyclin-dependent kinase inhibitor p27 have been observed in a range of malignancies, including lung cancer. To further elucidate the role of the molecule in tumor growth regulation, we evaluated p27 expression in a series of non-small cell lung carcinomas (NSCLCs), and examined its relation with histology, kinetic parameters, ploidy, and overall survival. We extended our investigation into the association of p27 levels with the presence of Ki-ras mutations, as well as with the expression status of p53 and pRb in tumor cells. MATERIAL AND METHODS: p27, p53, and pRb status were immunohistochemically evaluated in a total of 69 NSCLCs. In situ assays were employed to assess the kinetic parameters (Ki-67 immunohistochemistry for proliferation index, Tdt-mediated dUTP nick end labeling assay for apoptotic index). The ploidy status of the tumors was assessed after staining nuclei with the Feulgen procedure, and the presence of Ki-ras mutations was examined by restriction fragment length polymorphisms. All possible associations were assessed with a series of statistical methods. RESULTS: Immunoreactivity for p27 was observed in the entire series of specimens, with the mean percentage of positive cells being 33%. Adenocarcinomas (AdCs) exhibited higher p27 levels compared to squamous cell carcinomas (SqCCs) (p < 0.01). An inverse correlation was established between p27 expression and proliferation index (PI) (r = -0.834, p < 0.01) but not with apoptotic index (AI), whereas aneuploid tumors were characterized by lower p27 levels than diploid ones (p < 0.01). No difference in p27 immunostaining was observed with regard to the presence of Ki-ras mutations, whereas aberrant p53 and/or pRb expression patterns were associated with p27 underexpression (p < 0.01 for p53 status, p < 0.05 regarding pRb levels, and p < 0.01 for a combined deregulation of both proteins). Two or more alterations in the p27/p53/pRb protein network (i.e., p27 levels lower than the estimated mean value, overexpressed p53, and/or aberrant pRb) were associated with increased PI and aneuploidy (p < 0.001 and p < 0.01, respectively). A powerful trend was found between p27 expression and overall survival (p = 0.066). CONCLUSIONS: Our findings confirm the heterogeneity between AdCs and SqCCs, and are suggestive of an increased proliferative activity in NSCLCs underexpressing p27. Furthermore, our analysis supports the concept of p27 forming a functionally compact network with p53 and pRb, which is actively involved in the regulation of cellular proliferation and chromosomal stability.     

An immunohistochemical study of the expression of cell-cycle-regulated proteins p53, cyclin D1, RB, p27, Ki67 and MSH2 in gallbladder carcinoma and its precursor lesions

Gallbladder carcinomas are rare but highly lethal neoplasms. We examined the expression of five cell-cycle-related molecules (p53, RB, cyclin D1, p27, Ki-67), and MSH2, in 46 carcinomas, 14 adenomas, 15 low-grade dysplasias, 9 intestinal metaplasias and 20 normal gallbladder epithelia. The expression of these molecules was altered in gallbladder carcinomas and adenomas. In gallbladder carcinomas we observed increased expression of p53, cyclin D1, Ki-67, and MSH2 together with decreased expression of RB and p27 protein. Aberrant expression of cyclin D1 and reduced expression of RB were noted in adenomas, and expression of cyclin D1 was elevated in low-grade dysplasias. However, there was no change in the levels of these cell-cycle molecules in metaplasia. Expression of p53, p27, Ki-67, and MSH2 was correlated with clinical stage (P<0.05) and there was also a correlation between the expression of Ki-67 and MSH-2 and patient age (P<0.05). These results suggest that altered expression of cell-cycle molecules p53, cyclin D1, RB, p27, and of MSH-2 is involved in the progression of gallbladder carcinomas.     

Expression of Skp2 and p27KIP1 in naevi and malignant melanoma of the skin and its relation to clinical outcome

Skp2 (S-phase kinase associated protein 2) controls progression from G- to S-phase by promoting the proteolysis of the cyclin dependent kinase inhibitor p27KIP1. Despite the fact that a p27KIP1 decrease has been documented in melanoma progression, the role of Skp2 in these tumours is unknown. We therefore examined by immunohistochemistry the expression of Skp2, p27KIP1 and Ki-67 in 10 naevi (Ns), 15 superficial spreading melanomas (SSMs), 10 nodular melanomas (NMs) and 14 melanoma metastases (Ms). Nuclear Skp2 expression augmented with increasing malignancy (Ns: 1.4%, SSMs: 5.6%, NMs: 17.3%, Ms: 19.1%). In all tumours nuclear Skp2 expression correlated with Ki-67 (p=0.024) and inversely with p27KIP1 (p=0.007). A cytoplasmic reaction for Skp2 was also observed in most tumours and its expression decreased from Ns (12.3%) to SSMs (7.9%) and NMs (4.5%). In contrast, Ms showed an increase of cytoplasmic Skp2 (11.9%) that correlated with its nuclear expression (p=0.016). While nuclear Skp2 expression correlated with the pT-level (p=0.023), Clark-level (p=0.023) and Breslow index (p=0.019), the cytoplasmic Skp2 expression might be of biological significance only in NMs since it correlated with tumour depth (p=0.02) and pT-level (p=0.025). Our data suggests that Skp2 could contribute to melanoma progression. This is further highlighted by the fact that vertical growth phase (VGP) melanomas show significant higher nuclear Skp2 expressions when compared with the harmless radial growth phase (RGP) (p=0.047). Also nuclear Skp2 expression correlates with a reduced survival time (p=0.025) in melanoma.     

Linkage of Curcumin-Induced Cell Cycle Arrest and Apoptosis by Cyclin-Dependent Kinase Inhibitor p21/WAF1/CIP1

We have recently shown that curcumin induces apoptosis in prostate cancer cells through Bax translocation to mitochondria and caspase activation, and enhances the therapeutic potential of TRAIL. However, the molecular mechanisms by which it causes growth arrest are not well-understood. We studied the molecular mechanism of curcumin-induced cell cycle arrest in prostate cancer androgen-sensitive LNCaP and androgen-insensitive PC-3 cells. Treatment of both cell lines with curcumin resulted in cell cycle arrest at G1/S phase and that this cell cycle arrest is followed by the induction of apoptosis. Curcumin induced the expression of cyclin-dependent kinase (CDK) inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1, and inhibited the expression of cyclin E and cyclin D1, and hyperphosphorylation of retinoblastoma (Rb) protein. Lactacystin, an inhibitor of 26 proteasome, blocks curcumin-induced down-regulation of cyclin D1 and cyclin E proteins, suggesting their regulation at level of posttranslation. The suppression of cyclin D1 and cyclin E by curcumin may inhibit CDK-mediated phosphorylation of pRb protein. The inhibition of p21/WAF1/CIP1 by siRNA blocks curcumin-induced apoptosis, thus establishing a link between cell cycle and apoptosis. These effects of curcumin result in the proliferation arrest and disruption of cell cycle control leading to apoptosis. Our study suggests that curcumin can be developed as a chemopreventive agent for human prostate cancer.