5071

PARK2

AR-JP|LPRS2|PDJ|PRKN;parkin RBR E3 ubiquitin protein ligase;PARK2;parkin RBR E3 ubiquitin protein ligase

E3 ubiquitin-protein ligase parkin|Parkinson disease (autosomal recessive, juvenile) 2, parkin|parkinson juvenile disease protein 2|parkinson protein 2, E3 ubiquitin protein ligase (parkin)

6q25.2-q27

protein-coding

In an effort to identify tumor suppressor gene(s) associated with the frequent loss of heterozygosity observed on chromosome 6q25-q27, we constructed a contig derived from the sequences of bacterial artificial chromosomeP1 bacteriophage artificial chromosome clones defined by the genetic interval D6S1581-D6S1579-D6S305-D6S1599-D6S1008. Sequence analysis of this contig found it to contain eight known genes, including the complete genomic structure of the Parkin gene. Loss of heterozygosity (LOH) analysis of 40 malignant breast and ovarian tumors identified a common minimal region of loss, including the markers D6S305 (50%) and D6S1599 (32%). Both loci exhibited the highest frequencies of LOH in this study and are each located within the Parkin genomic structure. Whereas mutation analysis revealed no missense substitutions, expression of the Parkin gene appeared to be down-regulated or absent in the tumor biopsies and tumor cell lines examined. In addition, the identification of two truncating deletions in 3 of 20 ovarian tumor samples, as well as homozygous deletion of exon 2 in the lung adenocarcinoma cell lines Calu-3 and H-1573, supports the hypothesis that hemizygous or homozygous deletions are responsible for the abnormal expression of Parkin in these samples. These data suggest that the LOH observed at chromosome 6q25-q26 may contribute to the initiation andor progression of cancer by inactivating or reducing the expression of the Parkin gene. Because Parkin maps to FRA6E, one of the most active common fragile sites in the human genome, it represents another example of a large tumor suppressor gene, like FHIT and WWOX, located at a common fragile site.

The cloning and characterization of the common fragile site (CFS) FRA6E (6q26) identified Parkin, the gene involved in the pathogenesis of many cases of juvenile, early-onset and, rarely, late-onset Parkinsons disease, as the third large gene to be localized within a large CFS. Initial analyses of Parkin indicated that in addition to playing a role in Parkinsons disease, it might also be involved in the development and/or progression of ovarian cancer. These analyses also indicated striking similarities among the large CFS-locus genes: fragile histidine triad gene (FHIT; 3p14.2), WW domain-containing oxidoreductase gene (WWOX; 16q23), and Parkin (6q26). Analyses of FHIT and WWOX in a variety of different cancer types have identified the presence of alternative transcripts with whole exon deletions. Interestingly, various whole exon duplications and deletions have been identified for Parkin in juvenile and early-onset Parkinsons patients. Therefore, we performed mutational/exon rearrangement analysis of Parkin in ovarian cancer cell lines and primary tumors. Four (66.7%) cell lines and four (18.2%) primary tumors were identified as being heterozygous for the duplication or deletion of a Parkin exon. Additionally, three of 23 (13.0%) nonovarian tumor-derived cell lines were also identified as having a duplication or deletion of one or more Parkin exons. Analysis of Parkin protein expression with antibodies revealed that most of the ovarian cancer cell lines and primary tumors had diminished or absent Parkin expression. While functional analyses have not yet been performed for Parkin, these data suggest that like FHIT and WWOX, Parkin may represent a tumor suppressor gene.

PURPOSE: Parkin, a gene mutated in autosomal recessive juvenile Parkinsonism and mapped to the common fragile site FRA6E on human chromosome 6q25-q27, is associated with a frequent loss of heterozygosity and altered expression in breast and ovarian carcinomas. In addition, homozygous deletions of exon 2 creating deleterious truncations of the Parkin transcript were observed in the lung adenocarcinoma cell lines Calu-3 and H-1573, suggesting that the loss of this locus and the resulting changes in its expression are involved in the development of these tumors. EXPERIMENTAL DESIGN: We examined 20 paired normal and non-small cell lung cancer samples for the presence of Parkin alterations in the coding sequence and changes in gene expression. We also restored gene expression in the Parkin-deficient lung carcinoma cell line H460 by use of a recombinant lentivirus containing the wild-type Parkin cDNA. RESULTS: Loss of heterozygosity analysis identified a common region of loss in the Parkin/FRA6E locus with the highest frequency for the intragenic marker D6S1599 (45%), and semi-quantitative reverse transcription-PCR revealed reduced expression in 3 of 9 (33%) lung tumors. Although we did not observe any in vitro changes in cell proliferation or cell cycle, ectopic Parkin expression had the ability to reduce in vivo tumorigenicity in nude mice. CONCLUSION: These data suggest that Parkin is a tumor suppressor gene whose inactivation may play an important role in non-small cell lung cancer tumorigenesis.

The PARK2 gene, previously identified as a mutated target in patients with autosomal recessive juvenile parkinsonism (ARJP), has recently been found to be a candidate tumor suppressor gene in ovarian, breast, lung and hepatocellular carcinoma that maps to the third common fragile site (CFS) FRA6E. PARK2 is linked to a novel described PACRG gene by a bidirectional promoter containing a defined CpG island in its common promoter region. We have studied the role of promoter hypermethylation in the regulation of PARK2 and PACRG expression in different tumor cell lines and primary patient samples. Abnormal methylation of the common promoter of PARK2 and PACRG was observed in 26% of patients with acute lymphoblastic leukemia and 20% of patients with chronic myelogenous leukemia (CML) in lymphoid blast crisis, but not in ovarian, breast, lung, neuroblastoma, astrocytoma or colon cancer cells. Abnormal methylation resulted in downregulation of PARK2 and PACRG gene expression, while demethylation of ALL cells resulted in demethylation of the promoter and upregulation of PARK2 and PACRG expression. By FISH, we demonstrated that a lack of PARK2 and PACRG expression was due to biallelic hypermethylation and not to deletion of either PARK2 or PACRG in ALL. In conclusion, our results demonstrate for the first time that the candidate tumor suppressor genes PARK2 and PACRG are epigenetically regulated in human leukemia, suggesting that abnormal methylation and regulation of PARK2 and PACRG may play a role in the pathogenesis and development of this hematological neoplasm.

The parkin was first identified as a gene implicated in autosomal recessive juvenile Parkinsonism. Deregulation of the parkin gene, however, has been observed in various human cancers, suggesting that the parkin gene may be important in tumorigenesis. To gain insight into the physiologic role of parkin, we generated parkin-/- mice lacking exon 3 of the parkin gene. We demonstrated here that parkin-/- mice had enhanced hepatocyte proliferation and developed macroscopic hepatic tumors with the characteristics of hepatocellular carcinoma. Microarray analyses revealed that parkin deficiency caused the alteration of gene expression profiles in the liver. Among them, endogenous follistatin is commonly upregulated in both nontumorous and tumorous liver tissues of parkin-deficient mice. Parkin deficiency resulted in suppression of caspase activation and rendered hepatocytes resistant to apoptosis in a follistatin-dependent manner. These results suggested that parkin deficiency caused enhanced hepatocyte proliferation and resistance to apoptosis, resulting in hepatic tumor development, partially through the upregulation of endogenous follistatin. The finding that parkin-deficient mice are susceptible to hepatocarcinogenesis provided the first evidence showing that parkin is indeed a tumor suppressor gene.

mutation of the gene PARK2, which encodes an E3 ubiquitin ligase, is the most common cause of early-onset Parkinsons disease. In a search for multisite tumor suppressors, we identified PARK2 as a frequently targeted gene on chromosome 6q25.2-q27 in cancer. Here we describe inactivating somatic mutations and frequent intragenic deletions of PARK2 in human malignancies. The PARK2 mutations in cancer occur in the same domains, and sometimes at the same residues, as the germline mutations causing familial Parkinsons disease. cancer-specific mutations abrogate the growth-suppressive effects of the PARK2 protein. PARK2 mutations in cancer decrease PARK2s E3 ligase activity, compromising its ability to ubiquitinate cyclin E and resulting in mitotic instability. These data strongly point to PARK2 as a tumor suppressor on 6q25.2-q27. Thus, PARK2, a gene that causes neuronal dysfunction when mutated in the germline, may instead contribute to oncogenesis when altered in non-neuronal somatic cells.

Studies on loss of heterozygosity have been made for Parkin gene-specific microsatellite markers in malignancies like breast, ovary and lungs, and the results have shown a significant association. However, till date, there is no study with respect to Parkin gene-associated microsatellite markers in cervical cancer. The present study deals with the Parkin gene-associated microsatellite markers and the occurrence of its loss of heterozygosity in patients with human cervical cancer. DNA was isolated from the 105 cervical carcinoma samples and matched control specimens. Polymerase chain reaction was performed using primer specific for two intragenic markers D6S1599 and D6S305 present in Parkin introns 2 and 7, respectively, and one marker (D6S1008) at telomeric end and further electrophoresed on 8% denaturing polyacrylamide gel. Overall, 59 of 105 (56%) samples showed loss of heterozygosity in at least one locus in the region examined. The percentage of loss of heterozygosity for these markers ranged from 25% (D6S1008) to 48% (D6S305). Chi-square test was performed, and loss of heterozygosity was found significantly higher in both the intragenic markers (D6S1599 and D6S305) when compared with the locus at telomeric end (D6S1008) with P<0.05. These data argue that Parkin is a tumor suppressor gene whose inactivation may play an important role in the carcinoma of uterine cervix.