An Immunohistochemical Analysis of Cyclin D1, p53, and p21 Proteins in Tumors Originating fromthe Follicular Epithelium of the Thyroid Gland*

This study aimed at clarifying the factors closely related to the tumor progression of thyroid neoplasms. We examined the immunoreactivity of cyclin D1, p53, and p21^waf1/cip1 proteins in 179 thyroid tumors originating from the follicular epithelium using an immunohistochemical technique. Cyclin D1 positivity was frequent in well-differentiated thyroid carcinomas (39/122 cases), but it was rarely seen in follicular adenomas (1/33 cases), (p < 0.05). Positivity for p53 was more frequent in poorly differentiated carcinomas (7/19 cases) and undifferentiated carcinomas (4/5 cases) than in well-differentiated carcinomas (14/122 cases) (p < 0,05, respectively). P21^wafl/cip1 positivity was more frequent in well-differentiated thyroid carcinomas (43/122 cases) than in follicular adenomas (4/33 cases) (p < 0.05). Regarding the relationships of these proteins, co-positivity for cyclin D1 and p53 was observed more often in poorly differentiated carcinomas (5/7 cases) than in well-differentiated carcinomas (7/39 cases) (p < 0.05). Most cases with cyclin D1 positivity did not show p21^waf1/cip1 expression in poorly differentiated carcinomas (6/7 cases). Three cases examined showed co-positivity of p53 and p21^waf1/cip1.Our results suggest that cyclin D1 is invoved in thyroid oncogenesis. Moreover, p53 might be closely related to the development of poorly differentiated carcinomas and undifferentiated carcinomas originating from well-differentiated carcinomas.     

肾癌中cyclinD1和p27kip1的表达及其意义

目的探讨cyc lin D1、p27k ip1在普通型肾细胞癌(renal cell carc inom a,RCC)发生、发展中的作用。方法用半定量RT-PCR方法检测25例普通型RCC和10例肿瘤远端的正常肾组织中cyc lin D1的mRNA含量,用免疫组化方法检测76例普通型RCC中cyc lin D1和p27k ip1蛋白的表达,并对cyc lin D1、p27k ip1蛋白表达与临床病理参数的关系进行分析。结果普通型RCC中cyc lin D1的mRNA含量0.488±0.399,高于正常对照组0.089±0.066(P<0.01)。cyc lin D1的表达与肿瘤体积大小有关,体积大者cyc lin D1高表达(P<0.05)。普通型RCC中p27k ip1表达低于正常对照组,随着p27k ip1表达的降低,肿瘤的细胞核Fu-hrm an分级、TNM分期增高。结论cyc lin D1高表达和p27k ip1的低表达与普通型RCC的发生有关;p27k ip1的低表达可能促进肿瘤的演进,p27k ip1的表达可作为评价普通型RCC预后的参考指标。     

Expression of p21<Superscript>cip1</Superscript>, p27<Superscript>kip1</Superscript>, and p16<Superscript>INk4a</Superscript> Cyclin-Dependent Kinase Inhibitors in Papillary Thyroid Carcinoma: Correlation with Clinicopathological Factors

In a variety of human malignancies, aberrant expression of proteins involved in the control of cell-cycle progression has been reported. In this study, p21^cip1, p27^kip1, and p16^INk4a cyclin-dependent kinase inhibitors were analyzed to evaluate their usefulness in clinical management of papillary thyroid carcinoma (PTC). Archived material derived from 46 cases of PTC was analyzed immunohistochemically. Protein expression was ascertained on tissue microarrays, and results were correlated with clinicopathological features of the patients. Positive immunostaining was observed in 14 (30,4%) p21^cip1, 26 (56,5%) p27^kip1, and 14 (30,4%) p16^INk4a cases. No significant correlation between p21^cip1 or p27^kip1 and clinical factors was found. In contrast, p16^INk4a expression showed a significant correlation with initial extension of the disease. Therefore, 45.8% of patients with loco-regional extension were p16^INk4a positive, whereas overexpression was only seen in 15.7% of cases with intrathyroid disease (p < 0.05). Moreover, all patients with simultaneous p16^INk4a positivity and lack of p27^kip1 staining (four patients) presented lymph node metastases. In contrast, only 12 (28.5%) of the remaining patients showed lymph node tumor involvement. In conclusion, p16^INk4a expression suggests extrathyroid neck extension of PTC. This effect is enhanced when p27^kip1 is negative. We think that their analysis by immunohistochemistry could be useful in the management of patients with PTC.     

Overexpression of cyclin D1, D3, and p21 in an infantile renal carcinoma with Xp11.2 TFE3-gene fusion

Renal carcinomas harboring the TFE-3 translocation are rare and occur predominately in children and adolescents. Here, we report a case of infantile renal carcinoma with TFE3 translocation and show that the cell cycle is deregulated in this type of carcinoma. It is characterized by nuclear accumulation of cyclin D1 and D3 in combination with high levels of cyclin-dependent kinase inhibitor p21Cip1/Waf1 but without accumulation of p53, p16INK4a, or mdm2. The combined overexpression of p21, cyclin D1, and cyclin D3 was found exclusively in this type but not in other, more common types of renal carcinoma/oncocytoma (n=27). These results further underscore that renal carcinomas with Xp11. 2 translocations/TFE3-gene fusion represent a special type of renal neoplasm showing deregulation of specific cell cycle components. The analysis of further cases has to prove whether the derangement of the cell cycle is uniform and correlates with the specific type of molecular genetic derangement.     

Differential expression of cdk inhibitors p16, p21cip1, p27kip1, and cyclin E in cervical cytological smears prepared by the ThinPrep method

Our objective was to correlate p16, p21cip1, p27kip1, and cyclin E protein expression with the degree of dysplasia on ThinPrep Papanicolaou (Pap) smears using a modified immunoperoxidase staining. Smears read as normal, atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), or high-grade SIL (HSIL) were identified and tested for high-risk human papillomavirus (HR-HPV). Additional smears were processed for immunoperoxidase for p16, p21cip1, p27kip1, and cyclin E. Thirty-four smears were satisfactory for study. The p16 was positive in all nine HSIL, in four of nine LSIL, and in one of seven ASC-US. The p27kip1 was positive in all nine HSIL, in eight of nine LSIL, and in one of seven ASC-US. The p21cip1 was positive in all nine HSIL, in one of nine LSIL, and in one of seven ASC-US. Cyclin E was positive in seven of nine HSIL and in one of nine LSIL and in none of the ASC-US smears. Normal smears were negative for all the antigens. There was poor correlation of protein expression and HR-HPV infection. We concluded that p16, p21cip1, p27kip1, and cyclin E can be demonstrated on Pap smears and they are expressed differentially in dysplastic cells, with highest expression in HSIL. The p21cip1 and cyclin E showed the greatest correlation with HSIL.     

Genomic and immunophenotypical differences between hepatocellular carcinoma with and without cirrhosis

Tretiakova M S, Shabani‐Rad M T, Guggisberg K, Hart J, Anders R A & Gao Z‐h
(2010) Histopathology 56, 683–693
Genomic and immunophenotypical differences between hepatocellular carcinoma with and without cirrhosis Aims: To compare the expression of genes involved in p53, Wnt/β‐catenin, and retinoblastoma (Rb) 1 pathways between cirrhosis‐associated hepatocellular carcinoma (HCC‐C) and hepatocellular carcinoma arising in non‐cirrhotic liver (HCC‐NC). Methods and results: The gene expression profile was analysed using oligo‐DNA arrays, and then validated at protein level in a tissue microarray using immunohistochemistry. Compared with their background non‐neoplastic liver tissue, HCC‐C showed a significantly higher rate of p53, β‐catenin (protein only) and cyclin D1 expression, whereas HCC‐NC showed a significantly higher rate of p21^Waf1/cip1 and p27^Kip1 expression. HCC‐C had a significantly higher rate of p53 expression and a significantly lower rate of p21^waf1/cip1 expression than HCC‐NC. There was no statistically significant association between the expression of genetic markers and tumour histological grade, underlying aetiology, or lymphovascular invasion. Aberrant β‐catenin expression was more commonly seen in single tumours in comparison with multiple tumours. Increased p16^INK4 and p21^waf1/cip1 expression was more commonly observed in large‐sized tumours (>50 mm) than small‐sized tumours. Conclusions: Alteration of the p53 pathway plays a more important role in the pathogenesis of HCC‐C, whereas alterations in cell cycle regulators p21^waf1/cip1 and p27^Kip1 play a more important role in the pathogenesis of HCC‐NC.     

Expression of p27kip1 and p53 in medulloblastoma: relationship with cell proliferation and survival

p27kip1 and p21cip1 are cyclin-dependent kinase (cdk) inhibitors which along with p53 play critical roles in the control of cell cycle progression. Accumulation of p27kip1 in post-mitotic neurons is a major event of neurogenesis. We hypothesized that a dysregulation of the expression of p53 and these cdk inhibitors underlies cellular proliferation in medulloblastomas, and tested this hypothesis by investigating p27kip1, p21cip1, Bcl2 and p53 immunoreactivity in 14 medulloblastoma tumors. We noted an inverse relationship between p27kip1 expression and cellular proliferation (MIB1). Focal islands of neuroblastic or glial differentiation expressed high levels of p27kip1, while the undifferentiated, highly-proliferative population of tumor cells showed no detectable p27kip1 expression, thus suggesting a role for p27kip1 in cell cycle control in medulloblastoma. In addition, there was no detectable p21cip1 expression in any of the medulloblastomas studied. The low level of apoptosis displayed by these tumors was not associated with the expression of Bcl-2. A significant relationship was found between detection of p53 protein and poor survival. Since, p21cip1 and p27kip1 are often co-expressed with other INK4 family of cdk inhibitors during the induction of cellular differentiation and are synergistic in their effect, a deregulation of their coordinate expression may underlie the lack of complete differentiation in medulloblastoma.     

Regulation of DNA synthesis in mouse embryonic stem cells by transforming growth factor-α: Involvement of the PI3-K/Akt and Notch/Wnt signaling pathways

This study examined the mechanisms by which transforming growth factor (TGF)-α regulates proliferation of mouse embryonic stem (ES) cells. TGF-α increased [³H] thymidine and BrdU incorporation in a time- (0–72 h) and dose-dependent (0–10 ng/ml) manner. TGF-α stimulated the phosphorylation of Akt, mammalian target of rapamycin (mTOR), p70S6K1 and p44/42 mitogen-activated protein kinases (MAPKs). TGF-α also increased the protein levels of Notch, Notch intracellular domain, Hes-1 and Wnt1. However, TGF-α-induced DNA synthesis was blocked by inhibition of Akt, mTOR, p44/42 MAPKs and Notch. TGF-α increased the gene expression of c-jun, c-myc and c-fos. Moreover, TGF-α increased cyclin D/CDK 4 and cyclin E/CDK 2 levels, while decreasing p21^cip1/waf1 and p27^kip1, which were blocked by the inhibition of Akt, mTOR and Notch. In conclusion, TGF-α regulated DNA synthesis of mouse ES cells via PI3-K/Akt, p44/42 MAPKs and Notch/Wnt pathways.     

Recent classification of renal epithelial tumors

The recent classification of renal tumors is based on genetic evidence as well as on histologic features. Malignant tumor includes clear cell renal carcinoma (RCC), multilocular cystic RCC, papillary RCC, chromophobe RCC, carcinoma of the collecting duct of Bellini, renal carcinoma associated with Xp11.2 translocations/TFE3 gene fusions and mucinous tubular and spindle cell carcinoma. Benign tumor is subdivided into papillary adenoma, renal oncocytoma and metanephric adenoma. Recently, new disease entities such as acquired cystic disease-associated RCC, clear cell papillary RCC and renal carcinoma with t(6;11)(p21:q12) have been discovered. In this article, we briefly review and introduce the clinical, morphological and genetic features of these tumor entities.     

An immunohistochemical analysis of cyclin D1, p53, and p21waf1/cip1 proteins in tumors originating from the follicular epithelium of the thyroid gland

This study aimed at clarifying the factors closely related to the tumor progression of thyroid neoplasms. We examined the immunoreactivity of cyclin D1, p53, and p21waf1/cip1 proteins in 179 thyroid tumors originating from the follicular epithelium using an immunohistochemical technique. Cyclin D1 positivity was frequent in well-differentiated thyroid carcinomas (39/122 cases), but it was rarely seen in follicular adenomas (1/33 cases), (p < 0.05). Positivity for p53 was more frequent in poorly differentiated carcinomas (7/19 cases) and undifferentiated carcinomas (4/5 cases) than in well-differentiated carcinomas (14/122 cases) (p < 0,05, respectively). P21waf1/cip1 positivity was more frequent in well-differentiated thyroid carcinomas (43/122 cases) than in follicular adenomas (4/33 cases) (p < 0.05). Regarding the relationships of these proteins, co-positivity for cyclin D1 and p53 was observed more often in poorly differentiated carcinomas (5/7 cases) than in well-differentiated carcinomas (7/39 cases) (p < 0.05). Most cases with cyclin D1 positivity did not show p21waf1/cip1 expression in poorly differentiated carcinomas (6/7 cases). Three cases examined showed co-positivity of p53 and p21waf1/cip1. Our results suggest that cyclin D1 is invoved in thyroid oncogenesis. Moreover, p53 might be closely related to the development of poorly differentiated carcinomas and undifferentiated carcinomas originating from well-differentiated carcinomas.     

Cell Cycle Regulatory Proteins p27(kip), Cyclins Dl and E and Proliferative Activity in Oncocytic (Hurthle Cell) Lesions of the Thyroid

Cyclins are prime cell-cycle regulators central to the control of cell proliferation in eukaryotic cells. The formation of cyclin/cyclin-dependent kinases (CDK) complexes activates the kinases and initiates a cascade of events, which directs cells through the cell cycle. CDK inhibitors (CDKIs) such as p27^kip1 inhibit cyclin-CDK complexes and function as negative regulators of the cell cycle. Previous studies have shown that p27^kip1 is decreased in malignant relative to benign thyroid tumors, but its role and interaction with other cell cycle regulatory proteins have not been well established in oncocytic lesions of the thyroid. We studied the expression of p27^kip1, cyclins D1 and E, and Ki67 in 20 cases of oncocytic adenoma (AD), 6 cases of oncocytic carcinoma (CA), 8 cases of Hashimoto’s thyroiditis (HT), and 9 cases of nodular goiter with oncocytic change (NG) by immunohistochemistry. In the latter two lesions only oncocytic cells were evaluated. The positive staining was stratified into four groups. Statistical analysis was done using the Kruskal-Wallis one-way analysis of variance test, and, when significant, the Dunn multiple-comparisons procedure was used to determine pairwise differences. All 20 AD were p27^kip1 positive, 10 were 4+, 2 were 3+, and the remaining 8 were 1+. In contrast all 6 CA showed 4+ p27^kip1 staining, of the 8 HT, 2 were 4+, two 3+, three 1+, and 1 was negative. All 9 NG were p27 positive, 7 showed 4+, one 3+, and one 1+ staining. On pairwise comparison differences in p27^kip1 staining between AD and CA and between HT and CA were statistically significant (p=0.0243 and p=0.0142, respectively). In all but one case Ki67 expression was either very low (<3%) or negative. No significant differences were seen in the expression of cyclin D1 or cyclin E among the groups observed. In conclusion, the increased p27^kip1 expression in malignant oncocytic tumors relative to benign oncocytic lesions is unlike any other malignant progression reported in the thyroid and other organ systems in the body. This may reflect on the biologic nature of the oncocytic cells of the thyroid and the significance of this finding remains to be established. Proliferative activity as studied by Ki67 immunostaining was not helpful in distinguishing benign from malignant oncocytic tumors.     

A renal cell carcinoma with EWSR1‐TFE3 fusion gene

Both EWSR1 and TFE3 are well‐known oncogenes. EWSR1 encodes an RNA‐binding protein involved in multiple soft tissue tumors, including Ewing's sarcoma/peripheral neuroectodermal tumor, desmoplastic small round cell tumor, soft tissue clear cell sarcoma (malignant melanoma of soft parts), extraskeletal myxoid chondrosarcoma, and myxoid liposarcomas. TFE3 regulates both Golgi and lysosomal homeostasis and is rearranged in renal cell carcinoma (RCC), alveolar soft part sarcoma, epithelioid hemangioendothelioma, and perivascular epitheloid cell tumors (PEComas). In this report, we found a rare case of RCC with a fusion between 5′ EWSR1 and 3′ TFE3. The fusion product retained most functional motifs of TFE3. The oncogenic mechanism likely involves TFE3 overexpression through its juxtaposition with the regulatory elements of EWSR1 and its translocation to the nucleus, resulting in the deregulation of Golgi and lysosomal homeostasis. This is a second case of RCC containing EWSR1‐TFE3 fusion.     

Deregulated expression of cell cycle-associated proteins in solid pseudopapillary tumor of the pancreas.

Solid pseudopapillary tumor of the pancreas was studied in a 20-year-old woman and a 54-year-old woman. In the younger patient, the tumor had metastasized to the liver 8 years after distal pancreatectomy. In both neoplasms, the distinct histologic pattern of solid, pseudopapillary, and degenerative cystic areas was present. Analysis by means of immunohistochemistry revealed a diffuse expression for vimentin, neuron-specific enolase, and a focal positivity for al-antitrypsin, whereas epithelial markers were negative in the tumor of the older patient and only focally expressed in the tumor of the younger patient. Immunohistochemical analysis of cell cycle-associated proteins provided an overexpression of cyclin D1 and cyclin D3 in both tumors, although to varying degrees. In addition, the cyclin-dependent kinase inhibitors p21, and to a lesser extent p27, were up-regulated just as mdm2. There was no accumulation of p53 protein, and Ki67-positive cells were extremely scarce. Analysis of the liver metastases showed an immunoreactive profile similar to that of the primary tumor. The results show a deregulation of the cell cycle with overexpression of cell cycle-activating proteins D1 and D3 and a probably counterbalancing upregulation of the cyclin-dependent kinase inhibitors p21 and p27. The findings may explain the low pool of Ki67-reactive tumor cells and the generally good clinical outcome of these tumors. Whether a more profound dysbalance of the cell cycle regulation is responsible for the development of metastatic disease remains to be clarified.     

Alterations of expression of Rb,p16INK4A and cyclin D1 in non-small cell lung carcinoma and their clinical significance

Inactivation of the Rb pathway in non-small cell lung carcinoma (NSCLC) occurs mostly through inactivation of the cyclin-dependent kinase inhibitor p16^INK4A and/or up-regulation of cyclin D1. In order to assess the frequency and the prognostic value of these abnormalities in NSCLC, immunohistochemical analysis of Rb, p16^INK4, and cyclin D1 has been performed on 168 cases of NSCLC including 77 squamous cell carcinomas, 43 adenocarcinomas, and 48 basaloid carcinomas. The reduced survival rate of basaloid carcinoma (stage I–II) compared with other histological types of NSCLC was confirmed (p = 0·008). Loss of protein expression of Rb and p16^INK4A was observed in 12 per cent and 58 per cent of NSCLC cases respectively and cyclin D1 overexpression in 43 per cent. There was an inverse correlation between Rb and p16 expression ( p < 0·0001) and a direct correlation between Rb and cyclin D1 expression ( p = 0·0007). In univariate analysis, Rb-negative adenocarcinomas at stages I–II had a significantly shorter survival than Rb-positive cases ( p = 0·04) and stages I–II p16-positive cases had a shorter survival than p16-negative cases ( p = 0·02), which was more significant in basaloid carcinoma ( p = 0·003). p16 status retained its influence on survival in multivariate analysis at stage I–II for all cases ( p = 0·01) and for basaloid carcinoma ( p = 0·005). Cyclin D1 overexpression did not influence survival. Combined Rb/p16/cyclin D1 phenotypes in univariate analysis showed a shorter survival for Rb-negative/p16-positive/cyclin D1-negative tumours ( p = 0·002). These results, linked to previous data, indicate that the Rb pathway of G1 arrest is initially disrupted in the vast majority of NSCLCs (83 per cent), but could not confirm an unfavourable role for each individual event (p16^INK4A loss or cyclin D1 up-regulation) in prognosis. Copyright © 1999 John Wiley & Sons, Ltd.     

IGFBP7对人乳腺癌MCF-7细胞增殖的影响及其机制

目的: 探究胰岛素样生长因子结合蛋白7(IGFBP7)对人乳腺癌MCF-7细胞增殖的影响及其分子生物学机制。方法: 将质粒pCMV6-IGFBP7转染MCF-7细胞,构建稳定表达IGFBP7的MCF-7细胞系;采用Western blotting检测IGFBP7在MCF-7细胞稳定转染子的表达;采用软琼脂培养克隆形成实验检测IGFBP7对MCF-7细胞克隆形成能力的影响;采用流式细胞术检测IGFBP7对MCF-7细胞周期的影响;采用Western blotting检测IGFBP7对MCF-7细胞细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、细胞周期素D1(cyclin D1)、细胞周期素依赖性激酶4(CDK4)、cyclin E、CDK2、p21^CIP1/WAF1、p27^KIP1、p53、视网膜母细胞瘤蛋白(Rb)和p-Rb蛋白含量的影响。结果: (1)只有稳定转染质粒pCMV6-IGFBP7的MCF-7细胞表达IGFBP7。(2)IGFBP7能够显著降低MCF-7细胞的克隆形成率(P<0.01),阻止细胞从G₁期进入S 期,使其停滞于G₁期(P<0.01)。(3)IGFBP7能够显著抑制ERK1/2的磷酸化(P<0.01)。(4)IGFBP7能够下调cyclin D1和cyclin E蛋白表达(P<0.01),上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达(P<0.01),抑制Rb的磷酸化(P<0.01)。(5)MEK1/2阻断剂PD98059可部分模拟IGFBP7的肿瘤抑制效应。结论: (1) IGFBP7可通过下调cyclin D1和cyclin E蛋白表达,上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达,以及抑制Rb磷酸化发挥抗肿瘤作用;(2) IGFBP7对cyclin D1和p27^KIP1的调节可能与其抑制ERK1/2信号通路有关。     

p21WAF1/Cip1 is associated with cyclin D1CCND1 expression and tubular differentiation but is independent of p53 overexpression in human breast carcinoma

p21^WAF1/Cip1 is an inhibitor of cdk/cyclin complexes, and thus regulates the cell cycle. p21 is also related to cell differentiation and is regulated by wild-type p53, although p53-independent regulatory pathways have been proposed. In order to analyse p21 expression as well as its relationship with p53 in human breast cancer, an immunohistochemical analysis was undertaken of 77 breast carcinomas, 16 of them with an in situ component; 30 adjacent normal tissue samples; and five non-neoplastic specimens. Forty-four infiltrating carcinomas (57 per cent) were p21-positive. Expression of p21 was also observed in pre-invasive lesions, whereas normal ducts were negative or focally and weakly positive. p21 expression was associated with high histological grade (II+III) (P-0·017) and poor tubule formation (P-0·002), and was significantly less frequent in lobular carcinomas (P-0·0001). p21 positivity also correlated with increased proliferation, but this seemed to be dependent on the histological grade. Twenty carcinomas (26 per cent) showed p53 overexpression, but this was not associated with p21 negativity, suggesting the existence of p53-independent mechanisms for p21 regulation in vivo. Cyclin D1^CCND1 expression was analysed in the same series and an association between p21 and cyclin D1 expression was found, since 23 of 26 cyclin D1-positive carcinomas were p21-positive (P<0·001 …). In conclusion, p21 is frequently overexpressed in breast carcinomas and this occurs in the early stages of neoplastic progression. This overexpression seems to be independent of p53 status and might be involved in cyclin D1 modulation. © 1998 John Wiley & Sons, Ltd.