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Overexpression of CDC25A and CDC25B in Head and Neck Cancers' (CANCER RESEARCH57. 2366-2368. June 15, 19971 Advances in Brief Overexpression of CDC25A and CDC25B in Head and Neck Cancers' Daniela Gasparotto, Roberta Maestro, Sara Piccinin, Tamara Vukosavijevic, LUigi Barzan, Sandro Sulfaro, and Mauro Boiocchi2 Dis'ision of Experimental Oncology 1. (‘enirodi Riferimento Oncologico, via Pedemontana Occidentale 12. 33081 As'iano (PN) ID. G., R. M., S. P., T. V., M. B.), and Divisions of Otorhinolaryngology (L B.) and Pathology (S. S.). Pordenone City Hospital. 33/70 Pordenone. Italy Abstract inhibits kinase activity. These phosphorylation sites are conserved among eukaryotes, and their phosphorylation status is controlled by The deregulation of several cell cycle-related genes participates in the antagonistic action of wee! kinase and CDC25 phosphatases (4). neoplastic transformation. Cell cycle progression Is driven by cyclin In human cells, three CDC25 genes, CDC25A, CDC25B, and dependent kinases, which are positively regulated by association with cyclins and negatively regulated by binding to inhibitory subunits. The cDc25c, havebeenisolated(5—7).ThesethreeCDC25genesfunc activity of cydin.dependent kinases is also regulated by the phosphoryl tion at different phases of the cell cycle. CDC25A and CDC2SB are ation status, which is controlled by the antagonistic action of weel kinase expressed throughout the cell cycle with a peak expression in G@3—5 and CDC25 phosphatases. Three CDC25 genes are present in human cells: h after serum stimulation for CDC25A and in both G1-S-phase and G2 CDC25A, cDc2sB, and CDC25C. These three genes function at different for CDC25B (6, 8). CDC25C is predominantly expressed in G2 and phases of the cell cycle. Whereas CDC2SA and CDC2SB are expressed regulates the timing of entry into mitosis (7, 9). throughout the cell cycle, with peak expression in G, for CDC25A and in Several lines of evidence suggest a role for CDC25s as oncogenes. both G1-S-phase and G2 for CDC2SB, CDC25C is predominantly ex CDC25A and CDC25B have been shown to cooperate with either pressed in G2. Several lines of evidence suggest a role for CDC25s as mutated versions of Ha-ras or loss of Rbl in oncogenic transformation oncogenes. CDC25A and CDC2SB cooperate with Ha-ms or loss ofRbl in the oncogenic transformation of rodent fibroblasts. Moreover, they are of murine fibroblasts. The transfected cells can form colonies in soft transcriptional targets of c-myc, and CDC2SA in particular plays an agar and induce the formation of tumors when injected in nude mice important role as a mediator ofmyc functions. On the basis ofthe evidence (10).TheCDC25AandCDC25Bgenesaretranscriptionaltargetsof that CDC25 phosphatases can act as oncogenes, we analyzed the expres the c-myc oncogene, and CDC25A in particular plays an important sion of CDC2SA, CDC2SB, and CDC2SC genes In 20 squamous cell carci role as a mediator of myc-induced cell cycle activation as well as nomas of the head and neck by quantitative reverse transcription-PCR. apoptosis (1 1). Moreover, expression levels and phosphatase activity Our results show that whereas CDC2SC is expressed at a low level with no of CDC25A are strongly induced in fibroblasts by the adenovirus E1A relevant differences between neoplastic tissue and normal mucosa, oncoprotein, indicating that a high level of CDC25A presumably CDC2SA and CDC2SB are overexpressed in a large fraction of tumors. contributes to the ability of EIA to override the checkpoint control Introduction mechanisms during the G1 phase to induce DNA replication (12). Finally, CDC25A and CDC25B are overexpressed in some cancer cell Head and neck cancers are the sixth most common neoplasm in the lines (6) and in 32% of human breast cancers,4 suggesting a role for world (I). They include cancers of different sites, such as the larynx, these genes in neoplastic transformation. pharynx, oral cavity, and tongue, and over 90% of them are In this study, the role of CDC25 genes in head and neck cancer HNSCCs.3 Epidemiological surveys have shown tobacco smoking as development was analyzed by using a highly sensitive quantitative a major risk factor in the development of HNSCCs, but other factors, RT-PCR approach. Moderate or low levels of expression were de such as alcohol consumption and environmental exposure, play a tected for the CDC25C gene, with no relevant differences between synergistic role (2, 3). It is known that cancer arises as a consequence neoplastic tissue and normal mucosa. In contrast, CDC25A and of a series of genetic changes that are followed by clonal expansion of CDC25B were significantly overexpressed in a large fraction of cells through a selective growth advantage. The identification of the tumors, suggesting that a deregulated expression of these CDC25 nature and timing of these changes is critical for the biological phosphatases may play an important role in head and neck cancer understanding of the disease and can provide markers useful for development. diagnosis and prognosis. Accumulating evidence suggests that the deregulation of several Materials and Methods cell cycle-related genes participates in neoplastic transformation. In eukaryotes, cell cycle progression is controlled by a family of en Samples. Twenty squamous cell carcinomas and corresponding normal zymes known as CDKs (4). These enzymes consist of a catalytic mucosa of the upper aerodigestive tract were collected from patients at the City subunit (CDK) that is positively regulated by association with a Hospital of Pordenone. No patient had been treated with chemotherapy or regulatory subunit, known as cyclin, and negatively regulated by radiotherapy before surgery. All tissues were frozen in liquid nitrogen imme binding to inhibitory subunits (p16, p21, p27, and others). The activity diately after surgery and stored at —80°Cuntilextraction of RNA. of CDKs is also regulated by phosphorylation; inhibitory phosphoryl Quantitative PCR Analysis. Total cellular RNA was extracted using the ation of threonine and tyrosine residues in the ATP-binding site guanidinium thiocyanate method (13). Evaluation of gene expression by Northern blot analysis in fresh tumors is often prevented by the fact that insufficient amounts of RNA are recovered from these samples. In addition, Received 3/I 8/97; accepted 4/30/97. accurate analysis of genes expressed at a low level or of large transcripts, like The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with CDC25 mRNAs, requires very high-quality RNAs, because even a small 18 U.S.C. Section 1734 solely to indicate this fact. amount of degradation may impair the quantification of the transcript. To I Supported in part by a grant from the Italian Association for Cancer Research. circumvent these limitations, we adopted a very sensitive and quantitative 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; CDK. cyclin-dependent kinase; RT-PCR, reverse transcription-PCR. 4 K. Galaktionov, personal communication. 2366 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1997 American Association for Cancer Research. OVEREXPRESSIONOFCDC25AANDCDC25BIN HEADANDNECKCANCERS PCR-based analysis, competitive RT-PCR (14). In a competitive RT-PCR, a expressionCDC25ATable 2 CDC25 mRNA constantamountof reverse-transcribedRNAis mixedand coamplifiedwith expressionCase expression CDC25B expressionCDC25C fixed amounts of a synthetic competitor that differs from the target cDNA for the presence of a small insertion/deletion or a restriction site, such that the two mucosa)HN2(tumor/normal mucosa) (tumor/normal mucosa)(tumor/normal productsareeasilydistinguishablebygelelectrophoresis.Becauseofthe high 1.21.2HN4 1.5 similarity,the two productsare amplifiedwith the sameefficiency,and the 1.21HN5 4.5 2.20.8HN7 2.5 amount of target cDNA is quantified on the basis of the amount of synthetic 2.5IHNIO 5.5 competitor needed to produce an equivalent PCR signal. Differences in the 3.81.5HN16 3.5 amount of starting RNA as well as in reverse transcriptase efficiency in diverse 3.11.5HN18 5.1 samples are minimized by normalizing data in comparison with the expression 2.52HN2O 10 31.5HN28 5.4 of a housekeeping gene (in the present study, the ribosomal protein 514 gene). 1.51HN3O 3 also evaluated by competitive PCR. The system was standardized comparing 51.2H1N32 10 PCR and Northern blot data obtained from a series of head and neck-derived 3.81.5HN58 8 cell lines that showed different expression levels of CDC25 mRNAs (data not 2.50.7HNl39 12 3.4IHN158 3.6 shown). 5.51.5HN182 4.5 For RT-PCR, 1 @gofRNA was reverse-transcribed in a 20-pi reaction by 20.8HN189 5.2 random hexanucleotide priming using avian myeloblastosis virus reverse tran 2IHNI91 10 scriptase (Promega, Madison, WI) according to the manufacturer's instruc 41.2TC5O 7 1.41TC5I 1.2 tions. 3.51.5TC53 5.1 RNA expression analysis was carried out by a competitive PCR method 41.2Total 1.5 using synthetic competitors that differed from the cDNA of interest by having overexpressing―“Cases%o overexpressing― IO/@@ a small internal insertion. The primers used, the annealing temperature, and the thetumor were considered overexpressing when the ratio between expression in size of amplified fragments are reported in Table 1. The synthetic competitors and expression in normal tissue wa.s 3. were generated by overlap extension (15). The CDC25A synthetic competitor contained a l4-bp insertion between nucleotides 1425 and 1426 [the numera ies/p.g of RNA; CDC25B, (4.0 ±1.0) X 1O@'copies/pg of RNA; and tion is according to Galaktionov et aL (5)]. The competitor for CDC25B CDC25C, (5.0 ± I.2) x lO@copies/@.tg of RNA], and no relevant carried a 20-bp insertion between nucleotides 1266 and 1267 [the numeration difference in the expression level of CDC25A, CDC2SB, or CDC25C is according to Galaktionov et a!. (5)]. The competitor for CDC25C contained was detected among the different samples.
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