Changes in the interactions among proteins that participate in a biochemical pathway can reflect the immediate regulatory responses to intrinsic or extrinsic perturbations of the pathway. Thus, methods that allow for the direct detection of the dynamics of protein-protein interactions can be used to probe the effects of any perturbation on any pathway of interest. Here we describe experimental strategies - based on protein-fragment complementation assays (PCAs) - that can achieve this. PCA-based strategies can be used with or instead of traditional target-based drug discovery strategies to identify novel pathway-component proteins of therapeutic interest, to increase the quantity and quality of information about the actions of potential drugs, and to gain insight into the intricate networks that make up the molecular machinery of living cells.

Multiple chromosome 3p tumor suppressor genes (TSG) have been proposed in the pathogenesis of ovarian cancer based on complex patterns of 3p loss. To attain functional evidence in support of TSGs and identify candidate regions, we applied a chromosome transfer method involving cell fusions of the tumorigenic OV90 human ovarian cancer cell line, monoallelic for 3p and an irradiated mouse cell line containing a human chromosome 3 in order to derive OV90 hybrids containing normal 3p fragments. The resulting hybrids showed complete or incomplete suppression of tumorigenicity in nude mouse xenograft assays, and varied in their ability to form colonies in soft agarose and three-dimensional spheroids in a manner consistent with alteration of their in vivo tumorigenic phenotypes. Expression microarray analysis identified a set of common differentially expressed genes, such as SPARC, DAB2 and VEGF, some of which have been shown implicated in ovarian cancer. Genotyping assays revealed that they harbored normal 3p fragments, some of which overlapped candidate TSG regions (3p25-p26, 3p24 and 3p14-pcen) identified previously in loss of heterozygosity analyses of ovarian cancers. However, only the 3p12-pcen region was acquired in common by all hybrids where expression microarray analysis identified differentially expressed genes. The correlation of 3p12-pcen transfer and tumor suppression with a concerted reprogramming of the cellular transcriptome suggest that the putative TSG may have affected key underlying events in ovarian cancer.

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We have investigated previously the utility of oligonucleotide expression microarray technology in an analysis of four spontaneously transformed epithelial ovarian cancer (EOC) cell lines, TOV-21G, TOV-81D, OV-90, and TOV-112D. Here, we examine the expression of 290 expressed sequence tags (ESTs) that map to human chromosome 3 in a primary culture derived from normal ovarian surface epithelium (NOSE), NOV-31, and the four spontaneously transformed EOC cell lines. One of these cell lines, OV-90, harbors a deletion of an entire chromosome 3p arm. Whereas the most aggressive cell lines (OV-90, TOV-112D, and TOV-21G) exhibited the highest levels of expression, assessed by the mean of expression values of all ESTs, OV-90 showed the lowest mean of expression of ESTs that map to the 3p arm in comparison with TOV-112D and TOV-21G. This difference in expression profile of 3p ESTs in OV-90 is also reflected in the ratio of expression of ESTs on 3p versus the 3q arm and in that the expression values of ESTs that map to 3p were more often lower than higher in OV-90 in two-way comparisons with NOV-31, TOV-21G, and TOV-112D. The loss of a 3p arm does not affect the pattern of differential expression in analyses based on the range of numeric expression values of each EST or fold differences in expression for each EST in comparison with NOV-31. However, 25 differentially expressed ESTs were identified on the basis of threefold differences in expression values between NOV-31 and any EOC cell line; and six of these ESTs were differentially expressed uniquely in OV-90. The investigation of these ESTs could facilitate the identification of novel chromosome 3 genes implicated in ovarian tumorigenesis.

Loss of heterozygosity (LOH) analyses of epithelial ovarian cancers (EOCs) previously identified a candidate tumor suppressor gene (TSG) locus within the chromosomal region 3p25.3-pter. Loss of heterozygosity analysis was performed to define the locus and identify candidates for further study. Loss of heterozygosity analysis of 124 malignant EOC samples of different histopathologic subtypes using 12 polymorphic microsatellite repeat markers identified a 330-kilobase minimal region of overlapping deletions at 3p26.3 that contained contactin 4 (CNTN4) as the only known TSG candidate. However, evaluation of the LOH patterns in the serous EOC samples, the most common subtype, enabled the identification of a second, broader region of LOH also included the cell adhesion molecule with homology to L1CAM (CHL1) and CNTN6 as candidates. Gene expression by reverse transcription polymerase chain reaction was not detectable in primary cultures of normal ovarian surface epithelial cells for any of these candidates. CNTN6 expression was also not detectable in serous EOC samples. In contrast, gene expression of CNTN4 and CHL1, particularly overexpression of CHL1, was observed in serous EOC samples. Mutation and gene expression analyses of well-defined EOC cell lines (OV-90, TOV-112D, TOV-21G, and TOV-81D) that differ in their tumorigenic potential and chromosome 3p26-pter genomic content revealed CNTN4 expression and a novel mutation only in the tumorigenic EOC cell line TOV-21G. This mutation was neither observed in controls (n = 105) nor detected by sequencing analysis of complementary DNA. Taken together, these results do not support the candidacy of CHL1, CNTN6, and CNTN4 as TSGs in the 3p26-pter region. However, the overexpression of CHL1, a member of the L1 cell adhesion molecule (L1CAM) family, warrants further investigation.

We previously reported the identification of three minimal regions of deletion on the short arm of chromosome 3 (3p) in epithelial ovarian tumor specimens, suggesting that the inactivation of tumor-suppressor genes in these regions may be important in terms of ovarian tumorigenesis. Another previous study of ovarian cancer observed that allele loss of chromosome 179 was frequently found in ovarian tumors that also showed loss of heterozygosity (LOH) of chromosomes 3p, 13q, 17p, and Xp. In an independent study, we also reported a high frequency of LOH for selected chromosome 17 loci in high-grade and late-stage ovarian tumors. We have extended our LOH analysis of chromosome 3p to include 102 ovarian tumor specimens (29 and 73 samples were previously examined for LOH of chromosome 3p and 17 markers, respectively), using additional polymorphic markers, to assess the coordinate LOH of loci representing the three chromosome 3p minimal regions of deletions [von Hippel-Lindau syndrome (VHL), thyroid hormone receptor beta, and fragile histidine triad (FHIT)] and LOH of other important loci [tumor protein 53 (TP53), breast cancer 1 early onset (BRCA1), breast cancer 2 early onset, retinoblastoma 1, ornithine carbamoyltransferase, and androgen receptor] or somatic mutations in TP53. There was a significant association between LOH of any chromosome 3p marker and LOH of any chromosome 17 marker (P = 0.026). The frequency of LOH at the TP53 locus was higher in the group of samples that displayed LOH of a 3p marker (P = 0.019), as was the frequency of LOH at the BRCA1 locus (P = 0.014). LOH of chromosome 3p was noted in four specimens that did not display LOH of either the BRCA1 or the TP53 locus, indicating that LOH of these loci need not precede LOH of the chromosome 3p loci. We found a significant association between LOH of the VHL (3p25) locus and LOH of any chromosome 17 marker (P = 0.005), suggesting that there may be an important relationship, in the tumorigenesis of epithelial ovarian cancer, between a gene at 3p25 and a gene located on chromosome 17. Our results indicate that inactivation of p53 by somatic mutation is unlikely to be a prerequisite to chromosome 3p LOH, because we found no significant association between mutations in TP53 and LOH of the three chromosome 3p loci. The frequency of LOH at the FHIT locus at 3p14 increased significantly with advancing age at diagnosis (P = 0.018), as did the frequency of somatic TP53 mutations (P = 0.008).