Relationship of p21 (waf1/cip1) and differentiation in chondrosarcoma cells

The histological grade of chondrosarcoma correlates well with their clinical behavior and with the patient's survival duration. We have previously demonstrated that p21 was expressed in the hypertrophic chondrocytes of the growth plate. To assess the relationship of p21 (waf1/cip1) to cell differentiation in chondrosarcoma, we examined the p21 expression in 14 cases of chondrosarcoma immunohistochemically and the induction of p21 by insulin-like growth factor-I (IGF-I) during cell differentiation in SW1353 chondrosarcoma cells. p21 immunoreactivity was seen in well-differentiated chondrosarcoma cells and was mutually exclusive with MIB1 reactivity in grade-1 chondrosarcoma. In vitro, the proteoglycan synthesis of SW1353 cells was increased by IGF-I in a dose-dependent manner. However, cell proliferation was not markedly stimulated. Overexpressions of p21 mRNA and p21 protein in SW1353 cells were induced by IGF-I 100 ng/ml. Our results suggested that the p21 expression was directly related to tumor differentiation and that the p21 expression was an important mediator for IGF-I in chondrosarcoma cells.     

Cytogenetic and molecular genetic changes in malignant transformation of immortalized esophageal epithelial cells

The purpose of this study was to evaluate the extent to which the expression of p53, c-myc, bcl-2, ras genes and chromosomes, along with activity of hTERT, impacts on the malignant transformation of immortalized esophageal epithelial cells. The SHEE cell line was established from an embryonic esophageal epithelial cell induced by transduction of E6E7 genes of human papillomavirus type 18 (HPV18E6E7). In cells of the 85th passage (SHEE85), the malignant transformation of SHEE was confirmed by morphology, cell proliferative index and tumor formation in SCID mice. C-myc, p53, bcl-2 and ras genes were assayed by the multi-PCR method with house-keeping gene GAPDH as control. The modal number of chromosomes was analyzed and its expression of subunit of telomerase, hTERT, was assessed by RT-PCR. Expression of HPV18E6E7 was assayed by Western blotting. The results showed that cells of SHEE85 were atypical and exhibited proliferative status with a proliferation index of 45.70%. Tumors formed in SCID mice with invasion of adjacent tissue. The karyotype belonged to hypotriploid and displayed expression of hTERT. C-myc, k-ras, bcl-2 and p53 (expression of phosphoprotein) were positive in SHEE85. Expression of HPV18E6E7 was positive. Taken together, SHEE85 cells were in fully malignant transformation and their molecular mechanism involved the expression of cellular genes, such as p53, bcl-2, c-myc and ras, and aberrance of chromosomes. It is probable that all of these changes were related with HPV18E6E7.     

Dedifferentiated chondrosarcoma mimicking a giant cell tumor. Is this low grade dedifferentiated chondrosarcoma?

We report a very rare case of a dedifferentiated chondrosarcoma mimicking a benign giant cell tumor. A 22-year-old male was admitted to our hospital with a history of mild left wrist pain after a skiing trauma. Radiology revealed an extensive meta-epiphyseal osteolytic lesion in the distal ulna, which appeared to be a giant cell tumor. Histological examination showed a biphasic tumor comprising chondroid and non-chondroid areas with a giant cell-rich lesion resembling a conventional giant cell tumor of the bone. Immunohistochemistry showed no expression of p16^INK4a, VEGFR1, KDR (VEGFR2), VEGFR3, cKIT, MDM2 or CDK4. However, high expression of the tyrosine kinases PDGFRA and PDGFRB was observed. Molecular analysis showed no amplification of the cMYC gene and no activating mutations in the cKIT (exons 9 and 11) or PDGFRA (exon 18) genes.     

p53 expression in CMV-infected cells: association with the alternative expression of the p53 transactivated genes p21/WAF1 and MDM2

The p53 tumour suppressor gene is a cell cycle regulator, able to induce cell cycle arrest to allow DNA repair or apoptosis. The molecular mechanisms underlying p53 action imply transactivation of p53 dependent genes such as WAF1 (for wild type p53 associated fragment 1) and the murine double minute (MDM2) gene. In some cases, inactivation of the p53 gene results from p53 gene mutations leading to p53 protein accumulation, but in others it may results from mechanisms other than mutation, such as interaction with viral or cellular proteins. The expression of p53 protein and p53 transactivated gene proteins p21/WAF1 and MDM2, combined with in situ detection of apoptosis, was studied in specimens of CMV-infected patients as an in vivo model of p53 alteration not due to point mutation. p53 positivity was found in CMV + cells in different tissues, in cells with typical inclusion bodies, and in in situ hybridization and immunohistochemistry CMV + cells without inclusions (hidden infection). Although this p53 reactivity was accompanied by the expression of MDM2 and p21/WAF1 proteins, the patterns of MDM2 and p21/WAF1 protein expression were mutually exclusive, and were associated with the presence or absence of inclusion bodies. Nuclei bearing inclusion bodies were usually MDM2 +, p21/WAF1?, while hidden infected cells were usually MDM2?, p21/WAF1 +. Apoptosis was not detected in any tissue section from CMV-infected patients. Two alternative patterns were found in CMV-infected tissues: p53 +, p21/WAF1 +, MDM2?, or p53 +, p21/WAF1?, MDM2 + protein expression. These may represent examples of p53 dependent alternative effects in the course of CMV infection. Early stages are represented by CMV + cells without inclusion bodies, which display p53 and p21/WAF1 expression, suggesting that p53 could be acting as a growth suppressor protein. Late CMV infection is represented by cells harbouring inclusion bodies. These cells showed a p53 +, p21/WAF1?, MDM2 + profile, consistent with MDM2 mediated p53 inactivation. The absence of p21/WAF1 expression and lack of apoptosis suggest that the p53 protein expressed by MDM2 + cells could be functionally inactivated in CMV-infected cells with inclusion bodies. Previous studies have suggested that p53 inactivation by MDM2 over-expression occurs in sarcomas and lymphomas. Our observations seem to indicate that this mechanism of MDM2 mediated p53 inactivation may play a role in the late phase of CMV infection.     

High-resolution array comparative genomic hybridization analysis of human bronchial and salivary adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) is a rare but distinctive tumor. Oligonucleotide array comparative genomic hybridization has been applied for cataloging genomic copy number alterations (CNAs) in 17 frozen salivary or bronchial tumors. Only four whole chromosome CNAs were found, and most cases had 2-4 segmental CNAs. No high level amplification was observed. There were recurrent gains at 7p15.2, 17q21-25, and 22q11-13, and recurrent losses at 1p35, 6q22-25, 8q12-13, 9p21, 12q12-13, and 17p11-13. The minimal region of gain at 7p15.2 contained the HOXA cluster. The minimal common regions of deletions contained the CDKN2A/CDKN2B, TP53, and LIMA1 tumor suppressor genes. The recurrent deletion at 8q12.3-13.1 contained no straightforward tumor suppressor gene, but the MIRN124A2 microRNA gene, whose product regulates MMP2 and CDK6. Among unique CNAs, gains harbored CCND1, KIT/PDGFRA/KDR, MDM2, and JAK2. The CNAs involving CCND1, MDM2, KIT, CDKN2A/2B, and TP53 were validated by FISH and/or multiplex ligation-dependent probe amplification. Although most tumors overexpressed cyclin D1 compared with surrounding glands, the only case to overexpress MDM2 had the corresponding CNA. In conclusion, our report suggests that ACC is characterized by a relatively low level of structural complexity. Array CGH and immunohistochemical data implicate MDM2 as the oncogene targeted at 12q15. The gain at 4q12 warrants further exploration as it contains a cluster of receptor kinase genes (KIT/PDGFRA/KDR), whose products can be responsive to specific therapies.     

Genetic and epigenetic alterations in tumor progression in a dedifferentiated chondrosarcoma

In this case of a dedifferentiated chondrosarcoma, we searched for genetic or epigenetic alterations in both components of the tumor, the low grade chondroblastic component, and the high grade osteosacomatouscomponent. To date, only little is known about aberrant patterns of DNA methylation in chondrosarcomas. Microdissection was used as a valuable method for clearly separating the tissues. We examined CpG island methylation of 8 tumor suppressor genes and candidate tumor suppressor genes, which are involved in different pathways: cell cycle (p21WAF1, p16INK4, p14ARF), apoptosis (DAPK, FHIT), DNA repair (hMLH1), and cell adherence (E-Cadherin). We found p16INK4 and E-cadherin promotor methylation in the low grade chondroid compartment of the dedifferentiated chondrosarcoma. P16INK4, FHIT, and E-cadherin were methylated in the highly malignant osteosarcomatous compartment of the tumor. Earlier investigations of this chondrosarcoma showed p53 mutation and p53-LOH in the anaplastic component. As shown in this case, it was accompanied by Rb-LOH. Early methylation of p16IK4 and E-cadherin in the chondroid compartment could point to the monoclonal origin of demonstrated dedifferentiated chondrosarcoma. Further alterations, as shown in p53, Rb and FHIT, are responsible for the "switch" to a high grade anaplastic sarcoma.     

Overexpression of p21WAF1/CIP1 and MDM2 characterizes serous borderline ovarian tumors

Ovarian epithelial tumors are classically divided into benign, malignant, and borderline or of low malignant potential. It is controversial whether this last group of tumors should be considered benign or malignant. Expression of cell cycle markers has recently been linked to tumor behavior and response to treatment. It has been shown that one of the pathways through which the p53 gene controls the cell cycle is by transactivating p21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor. By inhibiting cdks, p21WAF1/CIP1 blocks the G-1 to S-phase transition in the cell cycle. p53 can be regulated by MDM2 (murine double minute-2) through direct inactivation or promotion of its cytoplasmic degradation. In an attempt to investigate the cell cycle checkpoint mechanisms of these tumors, we studied the expression of p53, Ki-67, MDM2, and p21WAF1/CIP1 by immunohistochemistry. We analyzed the expression of these proteins in 19 cystadenomas (8 serous and 11 mucinous), 40 borderline tumors (31 serous and 9 mucinous), and 18 serous carcinomas of the ovary. p21WAF1/CIP1 was expressed in 7 of 19 (37%) benign cystadenomas, 32 of 40 (80%) borderline tumors (93.5% of serous and 33% of mucinous), and in 9 of 18 (50%) serous carcinomas. Ki-67 was only weakly expressed in 8 of 19 (42%) benign cystadenomas, all borderline tumors showed Ki-67 staining in less than 50% of the cells, and 55% of serous carcinomas stained in more than 50% of tumor cells. p53 was absent in all but 1 of the cystadenomas, was expressed in 9 of 40 (22.5%) borderline tumors (25.8% of serous and 11% of mucinous), and in 10 of 18 (55%) carcinomas. All 11 implants of serous borderline tumors expressed p21WAF1/CIP1. Most serous borderline tumors expressed higher levels of MDM2 compared with the benign cystadenomas and carcinomas. Four of the serous borderline implants (40%) expressed MDM2. Coexpression of p21WAF1/CIP1 and MDM2 characterizes serous borderline tumors of the ovary and their implants, which suggests that these cell cycle control proteins are important in these tumors and may be related to tumor progression. Low expression of p53 protein in serous borderline tumors might be in part mediated by MDM2. This suggests that the p53 pathway is intact in most of these tumors, in contrast with carcinomas, in which high expression of p53 has been related to mutations of this gene.     

MDM2 expression is associated with progress of disease and WAF1 expression in resected lung cancer

Although MDM2, p21/WAF1, and p53 are considered as regulating each other based on in vitro studies, the relation in human lung cancer is not fully understood. The expressions of these proteins were examined immunohistochemically in 112 resected non-small cell lung cancer specimens and the correlation between them were analyzed. MDM2 was expressed in 45% of all lung cancers. In advanced stage, MDM2-positive cases were observed more frequently than in early stage, showing significant difference. No significant difference was observed in the prognosis of the patients regardless of the expression of any protein. Although no correlation was observed between MDM2 expression and p53 expression, or between p21/WAF1 expression and p53 expression, MDM2 expression was strongly related with p21/WAF1 expression. Therefore, MDM2 expression may relate to the progress of the stage of lung cancer, and MDM2 expression and p21/WAF1 expression may be associated not through the p53-related pathway.     

Molecular analysis of the 9p21 locus and p53 genes in Ewing family tumors.

SUMMARY: The EWS-ETS rearrangements, and their respective fusion gene products, are specifically associated with histopathologically Ewing family tumors (EFT). These translocations are implicated in generating malignant transformation of EFT, but the presence of additional genetic alterations must be considered in the pathogenesis of such tumors. We analyzed 26 samples (biopsies and/or nude mice xenotransplants) collected from 19 patients with an EFT to determine whether molecular and cytogenetic alterations of the G(1)/S checkpoint genes are implicated in the pathogenesis of EFT. We found inactivating p53 mutations in three (16%) cases, which correlated with a loss of p21(WAF1/Cip1) expression and with a monosomy of chromosome 17 in two cases. Homozygous deletion of the p16(INK4A)/p14(ARF) gene was detected in four (21%) cases, three with codeletion of the p15(INK4B) gene and with chromosome 9 abnormalities. In all of these cases, expression of the implicated genes was absent. Hypermethylation of the p16(INK4A) and p15(INK4B) genes was detected in two (10%) and three (16%) cases, respectively, and was correlated with a low level of gene expression. Neither cyclin D1, nor MDM2 and CDK4 amplification was observed. Kaplan-Meier analysis showed that patients with tumors carrying homozygous deletion of the 9p21 locus, or point mutations of the p53 gene, had poorer outcomes than those without these molecular alterations (p = 0.005). In conclusion, 58% (11 of 19) of the analyzed patients showed genetic or epigenetic alterations in either the 9p21 locus or p53 tumor suppressor genes, defining a subgroup of patients with poor clinical outcome. This fact points to an important role of the G(1)/S cell cycle checkpoint dysregulation in the pathogenesis of EFT.     

p21(Waf1/Cip1) expression and the p53/MDM2 feedback loop in gastric carcinogenesis

Data are non-existent regarding coincidental alterations in the expression of p53 and its downstream target genes MDM2 and p21(Waf1/Cip1) in gastric carcinogenesis. An immunohistochemical study was therefore performed to examine the interrelationships of p53, MDM2, and p21(Waf1/Cip1) expression in a series of Caucasian early gastric carcinomas and precursor lesions. In normal gastric mucosa, chronic gastritis, and intestinal metaplasia, the surface cells expressed p21(Waf1/Cip1) in the absence of detectable nuclear p53 and MDM2 protein. Nuclear p53 protein accumulation was found in 60 per cent of the carcinomas, with significant differences in staining characteristics between the Lauren types in the absence of detectable MDM2 protein ( p< 0.005). Nearly 80 per cent of the carcinomas expressed p21(Waf1/Cip1), irrespective of Lauren type. Stratification of the carcinomas according to histological grade and growth pattern did not result in significant differences in p53 and p21(Waf1/Cip1) expression. Finally, no significant correlation was found between overall p53 and p21(Waf1/Cip1) expression in early gastric carcinomas. It is concluded that p21(Waf1/Cip1) expression in the non-neoplastic mucosa most likely relates to cell senescence and/or terminal differentiation, perhaps even in a p53-independent manner. In view of p53/MDM2 homeostasis, the differences in p53 staining characteristics between intestinal and diffuse-type carcinomas probably result, at least in part, from a difference in the prevalence of p53 gene mutations. Moreover, p53-independent induction of p21(Waf1/Cip1) expression apparently occurs in a considerable proportion of early carcinomas. Finally, in contrast to other carcinomas, p21(Waf1/Cip1) expression is not significantly correlated with histological grade in gastric carcinomas, suggesting possible defects downstream of p21(Waf1/Cip1) as an underlying cause for this apparent discrepancy.     

骨肿瘤MDM2和p53基因的改变

目的从基因水平研究ＭＤＭ２和ｐ５３基因在骨肿瘤中的表达，探讨其在骨肿瘤发生发展中的作用。方法用地高辛标记原位杂交技术研究了３８例骨肿瘤（骨肉瘤１２例，软骨肉瘤１０例，骨巨细胞瘤１４例，软骨母细胞瘤２例）ＭＤＭ２和ｐ５３的表达情况，并分析两种基因表达之间的相互关系。结果ＭＤＭ２在骨肉瘤、软骨肉瘤和骨巨细胞瘤中的阳性率分别为４１．７％、５０．０％和３５．７％。ｐ５３的阳性率分别为５８．３％、４０．０％和２１．４％。２例软骨母细胞瘤ＭＤＭ２和ｐ５３均为阳性。ＭＤＭ２与ｐ５３基因表达呈显著正相关（Ｐ＜０．００５）。结论ＭＤＭ２和ｐ５３基因改变是骨肿瘤的一种常见现象，可能参与骨肿瘤的发生与发展。     

Integrated analysis of genome‐wide copy number alterations and gene expression in microsatellite stable,CpG island methylator phenotype‐negative colon cancer

Microsatellite stable (MSS), CpG island methylator phenotype (CIMP)‐negative colorectal tumors, the most prevalent molecular subtype of colorectal cancer, are associated with extensive copy number alteration (CNA) events and aneuploidy. We report on the identification of characteristic recurrent CNA (with frequency >25%) events and associated gene expression profiles for a total of 40 paired tumor and adjacent normal colon tissues using genome‐wide microarrays. We observed recurrent CNAs, namely gains at 1q, 7p, 7q, 8p12‐11, 8q, 12p13, 13q, 20p, 20q, Xp, and Xq and losses at 1p36, 1p31, 1p21, 4p15‐12, 4q12‐35, 5q21‐22, 6q26, 8p, 14q, 15q11‐12, 17p, 18p, 18q, 21q21‐22, and 22q. Within these genomic regions we identified 356 genes with significant differential expression (P < 0.0001 and ±1.5‐fold change) in the tumor compared to adjacent normal tissue. Gene ontology and pathway analyses indicated that many of these genes were involved in functional mechanisms that regulate cell cycle, cell death, and metabolism. An amplicon present in >70% of the tumor samples at 20q11‐20q13 contained several cancer‐related genes (AHCY, POFUT1, RPN2, TH1L, and PRPF6) that were upregulated and demonstrated a significant linear correlation (P < 0.05) for gene dosage and gene expression. Copy number loss at 8p, a CNA associated with adenocarcinoma and poor prognosis, was observed in >50% of the tumor samples and demonstrated a significant linear correlation for gene dosage and gene expression for two potential tumor suppressor genes, MTUS1 (8p22) and PPP2CB (8p12). The results from our integration analysis illustrate the complex relationship between genomic alterations and gene expression in colon cancer. © 2013 Wiley Periodicals, Inc.     

Noncorrelative c-myc and ras oncogene expression in squamous cell carcinoma cells with tumorigenic potential.

The distribution of heterogeneous cell types within human tumors was examined, and the biological behavior of tumors and different tumor cell lines was evaluated following implantation into surrogate hosts. In situ hybridization and immunohistochemistry were used to examine the expression of oncogenes and localization of the squamous cell carcinoma cell surface-associated antigens. Increased levels of H-ras mRNA and p21 protein were present in six tumors, but enhanced c-myc mRNA expression was observed in just two tumors. The distribution of oncogene mRNA and SCC antigen-positive cells was not uniform throughout the tumor. Isolation of cells from the tumors was accomplished by cell culture, growth in soft agar, and growth in the nude mouse. One nontumorigenic immortalized cell line, SCC-83-01-82, isolated by passage through soft agar, was treated with 50 micrograms/ml of methyl methane sulfonate (MMS). These MMS-converted cells subsequently expressed a tumorigenic phenotype. In situ hybridization of the tumors that developed in nude mice revealed increased c-myc and H-ras mRNA expression. Serial passage of the MMS-converted tumors in vivo was accompanied by consistent enhanced c-myc expression. However, the levels of H-ras and keratin mRNA expression decreased with passage in vitro. Northern blot analysis of c-myc and H-ras mRNA levels from the original SCC cell line showed no change in expression following MMS treatment. The data suggest that SCC-83-01-82 is a premalignant cell line established from a mixed cell population in the tumor mass. It can be converted to a malignant phenotype by treatment with MMS, and the persistence of malignancy is under molecular control other than changes in the level of c-myc and ras gene expression.     

Cytogenetic characterization of complex karyotypes in seven established melanoma cell lines by multiplex fluorescence in situ hybridization and DAPI banding

We report the use of multiplex fluorescence in situ hybridization (M-FISH) to resolve chromosomal aberrations in seven established melanoma cell lines with hypotriploid to hypertetraploid complex karyotypes. By simultaneous identification of all human chromosomes in single FISH experiments using a set of 52 directly labeled, whole chromosome painting probes, cryptic chromosomal translocations and the origin of unclear chromosomal material in structural rearranged and marker chromosomes could be identified, refining the tumor karyotypes in all seven cell lines. The number of structural aberrations in each cell line assigned with combined M-FISH and DAPI banding analysis ranged from 15 to 45. Altogether, 275 breakpoints could be assigned to defined chromosomal regions or bands. The chromosome arms 1p, 6q, 7p, 9p, and 11q which are known to be nonrandomly associated with melanoma tumorigenesis, were frequently involved in chromosomal breaks and/or copy number changes. This study also demonstrated the practical usefulness of combining M-FISH with conventional cytogenetic banding techniques for the characterization of complex tumor karyotypes with massive genomic alterations.     

Molecular analysis of p53, MDM2 and H-ras genes in low-grade central osteosarcoma

Low-grade central osteosarcoma is an uncommon form that is characterized by a long premorbid history, and is compatible with prolonged survival after treatment. However, molecular abnormalities are rare in low-grade central osteosarcomas, whereas p53 mutations occur in approximately 20% of conventional high-grade osteosarcomas. In this study, 21 cases of low-grade central osteosarcoma were analyzed for mutations of the p53 gene, amplification of the MDM2 gene, and mutations of the H-ras gene using formalin-fixed, paraffin-embedded materials. We also examined the expression of p53, MDM2, and p21WAF1 protein immunohistochemically and assessed the proliferation activities using the monoclonal antibody MIB-1. One case (4.7%) showed strong p53 immunoreactivity, whereas p53 gene mutations were not detected at all. Seven cases (33.3%) showed immunoreactivity for MDM2 protein. As for gene alterations, MDM2 amplification was found in four cases (19.0%). p21WAF1 expression was detected in 12 cases (57.1%). MIB-1-LI showed very low levels in all the cases and no significant correlation with p53 or MDM2 immuno-reactivity. None of the tumors showed H-ras mutations. In conclusion, the number of p53 gene alterations in low-grade central osteosarcomas is lower than that in conventional high-grade osteosarcomas. MDM2 alterations and p21WAF1 expression might be involved in the tumorigenesis of low-grade central osteosarcomas.