RESUMEN
Components of the pRb/p16/cyclin D1/CDK4 pathway are frequent targets in numerous tumour types, including those of pituitary origin. However, previous studies of pituitary tumours have examined individual components of this pathway. Therefore, to determine their overall contribution we have simultaneously examined the immunohistochemical status of pRb, p16 and cyclin D1 and analysed the CDK4 gene for a characterized activating mutation. Of the total pituitary tumour cohort (29 clinically non-functioning adenomas and 16 somatotrophinomas) abnormal expression of either pRb, p16 or cyclin D1 was observed in 36 of 45 (80%) tumours and was significantly (P = 0.005) associated with non-functioning tumours (27/29; 93%) compared with somatotrophinomas (9/16, 56%). Loss of either pRb or p16 expression was mutually exclusive in 23 of 45 (51%) tumours, whilst concomitant loss of pRb and p16 expression was observed in five tumours. Cyclin D1 overexpression was observed in 22 of 45 (49%) tumours, however, there was no significant association between overexpression of cyclin D1 and the expression status of either pRb or p16. In addition, no activating mutations within codon 24 of the CDK4 gene were detected. This study provides evidence for the first time that components of the pRb/p16/cyclin D1/CDK4 pathway, either alone or in combination, are frequently deregulated in human pituitary tumours, suggesting that this pathway may be a useful target in drug or gene therapeutic approaches.
Asunto(s)
Adenoma/metabolismo , Ciclina D1/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/genética , Fase G1 , Neoplasias Hipofisarias/metabolismo , Proteínas Proto-Oncogénicas , Proteína de Retinoblastoma/metabolismo , Fase S , Adenoma/genética , Adenoma/patología , Codón , Quinasa 4 Dependiente de la Ciclina , Cartilla de ADN , Humanos , Inmunohistoquímica , Mutación , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patologíaRESUMEN
Cyclin D1 plays an important role in the regulation of cell progression through G1 of the cell cycle and has been demonstrated to have oncogenic properties. Using RFLP-PCR, an A/G polymorphism within the cyclin D1 (CCND1) gene was analyzed in 151 sporadic human pituitary tumors, of which 60 were informative at this locus. Further analysis showed that in 15 of 60 (25%) tumors, there was evidence of allelic imbalance, which is indicative of gene amplification. Allelic imbalance was observed more frequently in invasive tumors (11 of 29 tumors; 38%) than in their noninvasive counterparts (4 of 31 tumors; 13%; P = 0.02). Forty-six of the tumors informative for the polymorphism were available for immunohistochemical analysis. Cyclin D1 expression (nuclear and/or cytoplasmic) was detected in 25 of 46 (54%) tumors. Of these cases, expression of nuclear cyclin D1 was detected in 9 of 46 (20%) tumors, whereas 16 of 46 (35%) tumors showed cyclin D1 staining exclusively confined to the cytoplasm. Neither nuclear staining nor cytoplasmic staining was observed in any of the normal pituitaries or in the negative control. Expression of cyclin D1 was observed in significantly more nonfunctional tumors (18 of 27 tumors; 67%) than in somatotrophinomas (7 of 19 tumors; 37%; P = 0.046). Nuclear cyclin D1 expression was observed more frequently in nonfunctional tumors (8 of 27 tumors; 30%) than in somatotrophinomas (1 of 19 tumors; 5%; P = 0.04). There was no correlation between cyclin D1 expression and tumor grade or between allelic imbalance of CCND1 and cyclin D1 expression. We conclude that amplification of CCND1 occurs in pituitary tumors and that the overexpression of cyclin D1 may be an early event in tumorigenesis. Cyclin D1 overexpression occurring in the absence of CCND1 allelic imbalance suggests that additional mechanisms responsible for deregulated cyclin D1 expression are involved in human pituitary tumorigenesis.
Asunto(s)
Adenoma/genética , Adenoma/metabolismo , Ciclina D1/biosíntesis , Ciclina D1/genética , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Alelos , Ciclo Celular , Amplificación de Genes , Hormona del Crecimiento/biosíntesis , Humanos , Inmunohistoquímica , Leucocitos/metabolismo , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Fracciones Subcelulares/metabolismoRESUMEN
Two recent studies have described allelic loss of an RB1 intragenic marker on chromosome 13q in aggressive and metastatic pituitary tumors that did not correlate with loss of pRB. The second report also showed that losses were more frequently associated with a more centromeric marker. Because both of these studies suggest the presence of another or other tumor suppressor genes (TSGs) on 13q, we carried out an allelotype analysis encompassing known and recently described TSG loci on 13q, together with immunohistochemical analysis of pRB. We analyzed 82 nonfunctional tumors and 53 somatotrophinomas subdivided into invasive and noninvasive cohorts. A significantly higher frequency of loss, at one or more of 13 markers, was evident in the invasive nonfunctional tumors (54%, 26 of 48) than in their noninvasive counterparts (29%, 10 of 34). An approximately equal frequency of loss was apparent in invasive (28%, 5 of 18) and noninvasive (31%, 11 of 35) somatotrophinomas at one or more markers. In those tumors harboring deletion, loss at two or more markers was more frequent in invasive nonfunctional tumors 65% (17 of 26) compared with 36% (4 of 11) of their noninvasive counterparts. In somatotrophinomas, 40% (2 of 5) of invasive tumors as compared with 64% (7 of 11) of noninvasive tumors had evidence of two or more deletions. In tumors showing loss at two or more loci, the majority showed large deletions; however, loss of the RB1 intragenic marker D13S153 was infrequent. In most cases, loss at individual markers was more frequent in invasive tumors than their noninvasive counterparts. A marker 3 cM telomeric to RB1 (D13S1319) showed the highest frequency of deletion in both invasive cohorts (29% of somatotrophinomas and 24% of nonfunctional tumors). Immunohistochemical analysis of pRB showed frequent loss in somatotrophinomas (27%, 9 of 33) in comparison with 4% (2 of 53) of non-functional tumors. Although loss of pRB did not correlate with loss of an intragenic marker or tumor grade, it was significantly associated with the somatotrophinoma subtype (P = 0.002). These data suggest that chromosome 13q is a frequent target for allelic deletion in pituitary tumors and point to another or other TSG loci in these regions.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 13 , Genes de Retinoblastoma , Neoplasias Hipofisarias/genética , Proteína de Retinoblastoma/análisis , Mapeo Cromosómico , Humanos , Inmunohistoquímica , Repeticiones de MicrosatéliteRESUMEN
The cyclin-dependent kinase inhibitor 2A/multiple tumor suppressor gene 1 (CDKN2A/MTS//p16) plays an important role in the control of progression from G to S-phase of the cell cycle through the inhibition of CDK4-mediated RBI phosphorylation. In this study we investigated 46 nonfunctional pituitary tumors and 21 somatotrophinomas for aberrant methylation of the CpG island contained within the CDKN2A gene as an alternative mechanism of gene silencing. We demonstrate methylation in 32/46 (70%) of nonfunctioning tumors, in contrast to 2/21 (9.5%) somatotrophinomas and 0/15 histologically normal postmortem pituitaries. Methylation in noninvasive and invasive nonfunctional tumors was approximately equal at 15/20 (75%) and 17/26 (65%), respectively. Immunohistochemical analysis showed an absence of CDKN2A protein in 25/32 (78%) methylated nonfunctioning tumors, demonstrating a highly significant overall correlation (P = 0.00007) between hypermethylation of the gene and absence of the p 16 protein. The association between hypermethylation and absence of CDKN2A protein remained when the cohort of nonfunctional tumors was further subdivided into noninvasive 12/15 (80%; P = 0.004) and invasive 13/17 (76%; P = 0.01), suggesting this to be an early event in pituitary tumorigenesis. In contrast, a single invasive methylated somatotrophinoma failed to express the CDKN2A protein. These data show that hypermethylation of the CpG island within exon 1, but not exon 2, of the CDKN2A gene is frequently associated with loss of protein expression in nonfunctional pituitary tumors, but not somatotrophinomas, suggesting different tumorigenic pathways.
Asunto(s)
Adenoma/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Neoplasias Hipofisarias/genética , Adenoma/química , Adenoma/metabolismo , Southern Blotting , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , ADN de Neoplasias/análisis , Hormona del Crecimiento/metabolismo , Humanos , Sueros Inmunes , Inmunohistoquímica , Neoplasias Hipofisarias/química , Neoplasias Hipofisarias/metabolismo , Análisis de Secuencia de ADNRESUMEN
We have screened 57 cases of primary, nonfunctional, pituitary adenomas for loss of heterozygosity of markers on chromosome 9p. Using a panel of 11 microsatellite markers, we found hemizygous deletion with at least one of the markers in 18 tumors (31.5%). The frequency of loss was similar in both noninvasive (8 of 26; 31%) and invasive tumors (10 of 31; 32%), suggesting that loss on this chromosome might be an early event in pituitary tumorigenesis. Two discrete areas of loss were punctuated by a region of retention of heterozygosity between the markers D9S171 and IFNA, indicative of homozygous deletion. However, multiplex PCR analysis (MTS1 and MTS2) and the presence of a 3' untranslated region polymorphism in MTS1 suggested that neither of these tumor suppressor genes was homozygously deleted. In 6 of the 18 tumors showing LOH, sufficient DNA was also available for Southern blot analysis and, in all cases, showed retention of MTS1. Cell mixing experiments of tumor cell DNA homozygously deleted for MTS1 with DNA in which neither copy of the gene was deleted only gave rise to a signal at contamination levels greater than 30% and could discriminate homozygous and hemizygous loss. These studies support the recent findings that mechanisms other than hemi- and homozygous deletion are most likely responsible for the loss of MTS1 gene product in pituitary tumors (M. Woloschak et al., Cancer Res., 56: 2493-2486, 1996.). These data show that losses on either side of 9p21-22, both or either of which may be deleted, are involved in pituitary tumorigenesis and provide evidence for distinct suppressor gene loci, in addition to MTS1, on chromosome 9p.