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Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells.

Kunnev D, Ivanov I, Ionov Y - BMC Cancer (2009)

Bottom Line: Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively.Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. Dimiter.Kunnev@Roswellpark.org

ABSTRACT

Background: Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. An increase in transcription in response to NMD inhibition of a subset of genes is a major cause of false positives when genes are selected for sequencing analysis. To distinguish between mRNA accumulations caused by stress response-induced transcription and nonsense-containing mRNA stabilizations is a challenge in identifying mutant genes using GINI.

Methods: To identify potential tumor-suppressor genes mutated in prostate cancer cell lines, we applied a version of GINI that involves inhibition of NMD in two steps. In the first step, NMD is inhibited in duplicate tissue-culture plates. During this step, both the substrate for NMD and stress-response mRNA transcripts are accumulated in cells. In the second step, transcription is inhibited in both plates and NMD is inhibited in one plate and released in the second plate. Microarray analysis of gene-expression profiles in both plates after the second step detects only the differences in mRNA degradation but not in mRNA accumulation.

Results: Analyzing gene expression profile alterations in 22RV1 and LNCaP prostate cancer cells following NMD inhibition we selected candidates for sequencing analysis in both cell lines. Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively. Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.

Conclusion: The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.

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Heterozygous bi-allelic inactivating mutations in PARD3 and AS3 genes in the LOH -free regions in LNCaP cells. (A). RT-PCR analysis shows the lower mRNA levels in the untreated and the increased levels in caffeine treated LNCaP and 22Rv1 cells for the PARD3 and for AS3 genes, respectively. (B). Sequencing chromatograms show heterozygous deletion of a T in the (T)5 coding repeat in one allele and C to T substitution resulting in the TAA stop codon in the other allele of the PARD3 gene in LNCaP cells (top); heterozygous deletion of a C in the (C)7 repeat in one allele and of an A in the (A)9 repeat in the other allele of the AS3 gene in the 22Rv1 cells (middle); and a homozygous deletion of a C in the (C)2 repeat of the CLPTM1 gene in the LNCaP cells (bottom). (B). Array CGH analysis shows normal genomic content in the PARD3 and AS3 loci in the LNCaP and 22Rv1 cells and the loss of heterozygosity for the CLPTM1 locus in LNCaP cells.
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Figure 1: Heterozygous bi-allelic inactivating mutations in PARD3 and AS3 genes in the LOH -free regions in LNCaP cells. (A). RT-PCR analysis shows the lower mRNA levels in the untreated and the increased levels in caffeine treated LNCaP and 22Rv1 cells for the PARD3 and for AS3 genes, respectively. (B). Sequencing chromatograms show heterozygous deletion of a T in the (T)5 coding repeat in one allele and C to T substitution resulting in the TAA stop codon in the other allele of the PARD3 gene in LNCaP cells (top); heterozygous deletion of a C in the (C)7 repeat in one allele and of an A in the (A)9 repeat in the other allele of the AS3 gene in the 22Rv1 cells (middle); and a homozygous deletion of a C in the (C)2 repeat of the CLPTM1 gene in the LNCaP cells (bottom). (B). Array CGH analysis shows normal genomic content in the PARD3 and AS3 loci in the LNCaP and 22Rv1 cells and the loss of heterozygosity for the CLPTM1 locus in LNCaP cells.

Mentions: Analyzing the quintile normalized, log-transformed Affymetrix hybridization data we identified 7 and 5 hybridization probes for the LNCap and 22Rv1 cells respectively that satisfied the chosen cut-off thresholds (Table 1). RT-PCR analysis (Figure 1A) shows that PARD3 and AS3 mRNA levels were lower in the LNCaP and in 22Rv1 cells respectively before NMD inhibition and were up-regulated in these cells following NMD inhibition with caffeine. By sequencing the cDNA corresponding to the identified probes, we found mutations in the PARD3 and AS3 genes in the LNCaP and 22Rv1 cells respectively. The homozygous frameshift mutation in another selected candidate in the LNCaP cells, in the cleft lip and palate-associated transmembrane protein 1 (CLPTM1), has been identified previously [13]. Figure 1 shows that inactivation of the PARD3 and AS3 genes in the LNCaP and 22Rv1 cells occurred through two independent mutations in both alleles of genes located in the regions of the cell genome identified by aCGH analysis as LOH-free (Figure 1C). The inactivation of the CLPTM1 gene in LNCaP cells on the other hand, most likely had occurred by mutation in one allele and the loss of the other as suggested by the gene's location in the region of LOH (Figure 1C). This result illustrates that combining GINI and aCGH helps to narrow down the number of candidates for sequencing but may result in missing genes inactivated by two independent mutations. It also shows the capability of the GINI to efficiently identify mutant genes regardless of the information on LOH.


Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells.

Kunnev D, Ivanov I, Ionov Y - BMC Cancer (2009)

Heterozygous bi-allelic inactivating mutations in PARD3 and AS3 genes in the LOH -free regions in LNCaP cells. (A). RT-PCR analysis shows the lower mRNA levels in the untreated and the increased levels in caffeine treated LNCaP and 22Rv1 cells for the PARD3 and for AS3 genes, respectively. (B). Sequencing chromatograms show heterozygous deletion of a T in the (T)5 coding repeat in one allele and C to T substitution resulting in the TAA stop codon in the other allele of the PARD3 gene in LNCaP cells (top); heterozygous deletion of a C in the (C)7 repeat in one allele and of an A in the (A)9 repeat in the other allele of the AS3 gene in the 22Rv1 cells (middle); and a homozygous deletion of a C in the (C)2 repeat of the CLPTM1 gene in the LNCaP cells (bottom). (B). Array CGH analysis shows normal genomic content in the PARD3 and AS3 loci in the LNCaP and 22Rv1 cells and the loss of heterozygosity for the CLPTM1 locus in LNCaP cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Heterozygous bi-allelic inactivating mutations in PARD3 and AS3 genes in the LOH -free regions in LNCaP cells. (A). RT-PCR analysis shows the lower mRNA levels in the untreated and the increased levels in caffeine treated LNCaP and 22Rv1 cells for the PARD3 and for AS3 genes, respectively. (B). Sequencing chromatograms show heterozygous deletion of a T in the (T)5 coding repeat in one allele and C to T substitution resulting in the TAA stop codon in the other allele of the PARD3 gene in LNCaP cells (top); heterozygous deletion of a C in the (C)7 repeat in one allele and of an A in the (A)9 repeat in the other allele of the AS3 gene in the 22Rv1 cells (middle); and a homozygous deletion of a C in the (C)2 repeat of the CLPTM1 gene in the LNCaP cells (bottom). (B). Array CGH analysis shows normal genomic content in the PARD3 and AS3 loci in the LNCaP and 22Rv1 cells and the loss of heterozygosity for the CLPTM1 locus in LNCaP cells.
Mentions: Analyzing the quintile normalized, log-transformed Affymetrix hybridization data we identified 7 and 5 hybridization probes for the LNCap and 22Rv1 cells respectively that satisfied the chosen cut-off thresholds (Table 1). RT-PCR analysis (Figure 1A) shows that PARD3 and AS3 mRNA levels were lower in the LNCaP and in 22Rv1 cells respectively before NMD inhibition and were up-regulated in these cells following NMD inhibition with caffeine. By sequencing the cDNA corresponding to the identified probes, we found mutations in the PARD3 and AS3 genes in the LNCaP and 22Rv1 cells respectively. The homozygous frameshift mutation in another selected candidate in the LNCaP cells, in the cleft lip and palate-associated transmembrane protein 1 (CLPTM1), has been identified previously [13]. Figure 1 shows that inactivation of the PARD3 and AS3 genes in the LNCaP and 22Rv1 cells occurred through two independent mutations in both alleles of genes located in the regions of the cell genome identified by aCGH analysis as LOH-free (Figure 1C). The inactivation of the CLPTM1 gene in LNCaP cells on the other hand, most likely had occurred by mutation in one allele and the loss of the other as suggested by the gene's location in the region of LOH (Figure 1C). This result illustrates that combining GINI and aCGH helps to narrow down the number of candidates for sequencing but may result in missing genes inactivated by two independent mutations. It also shows the capability of the GINI to efficiently identify mutant genes regardless of the information on LOH.

Bottom Line: Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively.Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. Dimiter.Kunnev@Roswellpark.org

ABSTRACT

Background: Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. An increase in transcription in response to NMD inhibition of a subset of genes is a major cause of false positives when genes are selected for sequencing analysis. To distinguish between mRNA accumulations caused by stress response-induced transcription and nonsense-containing mRNA stabilizations is a challenge in identifying mutant genes using GINI.

Methods: To identify potential tumor-suppressor genes mutated in prostate cancer cell lines, we applied a version of GINI that involves inhibition of NMD in two steps. In the first step, NMD is inhibited in duplicate tissue-culture plates. During this step, both the substrate for NMD and stress-response mRNA transcripts are accumulated in cells. In the second step, transcription is inhibited in both plates and NMD is inhibited in one plate and released in the second plate. Microarray analysis of gene-expression profiles in both plates after the second step detects only the differences in mRNA degradation but not in mRNA accumulation.

Results: Analyzing gene expression profile alterations in 22RV1 and LNCaP prostate cancer cells following NMD inhibition we selected candidates for sequencing analysis in both cell lines. Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively. Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.

Conclusion: The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.

Show MeSH
Related in: MedlinePlus