Parkin, a gene implicated in autosomal recessive juvenile parkinsonism, is a candidate tumor suppressor gene on chromosome 6q25-q27

Abstract

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.

Figure 1

Physical map of the D6S1581–D6S1008 genomic interval. (a) All microsatellite markers and bacterial artificial chromosome/P1 bacteriophage artificial chromosome clone sequences were obtained from the National Center for Biotechnology Information database. The positions and alignment of known genes identified in this region and the markers used for LOH analysis are indicated. The Parkin genomic structure according to ref. is also shown (Lower). (b) Summary of allelic loss in six breast and four ovarian malignant tumors with partial deletions at 6q25–q27. Each vertical line represents a single case and black, gray, and white circles represent LOH, retention of heterozygosity, and noninformative results, respectively. The shared minimal region of loss among informative cases is indicated on the right side between markers D6S1599 and D6S305.

Figure 2

Northern and Western analysis of Parkin expression in normal human tissues and tumor-derived cell lines. (a and b) Multiple normal tissue blots (CLONTECH; a) and poly(A)⁺ RNA (b) from several tumor-derived cell lines were hybridized with a 1.5-kb cDNA probe containing the entire Parkin ORF. (c) Western blot analysis of Parkin protein levels in tumor-derived cell lines. (d) Parkin expression analysis in malignant ovarian and breast tumors by semiquantitative RT-PCR. Representative results are shown for each.

Figure 3

Identification of tumor-specific Parkin transcripts. (a) PCR amplification of tumor and normal ovarian cDNA. Samples UPN 66 and UPN 223 possess both wild-type and aberrant transcripts, whereas UPN 323 and normal ovaries 1 and 2 possess only wild-type Parkin. (b) Sequence analysis of aberrant Parkin transcripts. A schematic representation of each variant is shown, along with the resulting truncated protein isoform. Also indicated is the location of primer pairs used for both analyses.

Figure 4

Analysis and mapping of HDs in the Parkin locus. (a) PCR analysis of exons 1–3 in Calu-3, H-1573, and H-460 cell lines and in normal human genomic DNA. (b) Southern analysis of Parkin gene in DNA digested with EcoRI or BglII. Blots were hybridized with either a Parkin exon 2-specific probe (Upper) or the full-length Parkin cDNA (Lower). Arrows indicate the absence of the expected 15.3- and 6.5-kb EcoRI and BglII fragments, respectively. PBL, human peripheral blood leukocyte. (c) A schematic representation of exon 2 deletions observed in the lung adenocarcinoma cell lines, Calu-3 and H-1573.