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Functional characterization of CLPTM1L as a lung cancer risk candidate gene in the 5p15.33 locus.

James MA, Wen W, Wang Y, Byers LA, Heymach JV, Coombes KR, Girard L, Minna J, You M - PLoS ONE (2012)

Bottom Line: This locus has been found by multiple genome wide association studies to be associated with lung cancer in both smokers and non-smokers.Bcl-xL accumulation was significantly decreased upon loss of CLPTM1L.Thus, this study implicates anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis and resistance to chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: MCW Cancer Center, Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
Cleft Lip and Palate Transmembrane Protein 1-Like (CLPTM1L), resides in a region of chromosome 5 for which copy number gain has been found to be the most frequent genetic event in the early stages of non-small cell lung cancer (NSCLC). This locus has been found by multiple genome wide association studies to be associated with lung cancer in both smokers and non-smokers. CLPTM1L has been identified as an overexpressed protein in human ovarian tumor cell lines that are resistant to cisplatin, which is the only insight thus far into the function of CLPTM1L. Here we find CLPTM1L expression to be increased in lung adenocarcinomas compared to matched normal lung tissues and in lung tumor cell lines by mechanisms not exclusive to copy number gain. Upon loss of CLPTM1L accumulation in lung tumor cells, cisplatin and camptothecin induced apoptosis were increased in direct proportion to the level of CLPTM1L knockdown. Bcl-xL accumulation was significantly decreased upon loss of CLPTM1L. Expression of exogenous Bcl-xL abolished sensitization to apoptotic killing with CLPTM1L knockdown. These results demonstrate that CLPTM1L, an overexpressed protein in lung tumor cells, protects from genotoxic stress induced apoptosis through regulation of Bcl-xL. Thus, this study implicates anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis and resistance to chemotherapy.

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DNA damage induced apoptosis is regulated by CLPTM1L.A) Annexin V binding by flow cytometry of A549 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. B) Relative caspase 3/7 activity in H838 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. Error bars represent one standard deviation from the mean. **- p<0.01 * - p<0.02 by two-tailed Student’s T-Test. C) Micrographs of A549 cells with CLPTM1L knockdown after 24 hours treatment with 50µM cisplatin showing increased genotoxic cell death upon loss of CLPTM1L.
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pone-0036116-g003: DNA damage induced apoptosis is regulated by CLPTM1L.A) Annexin V binding by flow cytometry of A549 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. B) Relative caspase 3/7 activity in H838 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. Error bars represent one standard deviation from the mean. **- p<0.01 * - p<0.02 by two-tailed Student’s T-Test. C) Micrographs of A549 cells with CLPTM1L knockdown after 24 hours treatment with 50µM cisplatin showing increased genotoxic cell death upon loss of CLPTM1L.

Mentions: Annexin V immunofluorescence detected by flow cytometry was used as a marker of early apoptosis in A549 cells with or without CLPTM1L knockdown and cisplatin treatment. We observed a dose dependent increase in apoptosis with loss of CLPTM1L (Figure 3A). While only 16% of cells with vector alone were apoptotic after 10µM cisplatin treatment for 24 hours, 76% of cells with shCLPTM1L-3 were apoptotic. Resistance to cisplatin induced apoptosis was found to be proportional to the amount of CLPTM1L transcript accumulation in these cells, with an r2 of 0.9808 (p = 2×10−8) between percent knockdown and percent apoptotic cells (Figure S3A). Cisplatin concentrations at the lower limits of sensitivity were used to allow resolution of sensitivities conferred by different levels of CLPTM1L knockdown. The DNA damaging agents cisplatin and nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) both caused accumulation of DNA strand breaks independently of CLPTM1L expression as detected by the COMET method (Figure S4). Therefore, the observed increase in sensitivity tositivity to cisplatin upon loss of CLPTM1L is due to an effect on apoptosis or apoptotic signaling rather than an effect on levels of acute DNA damage. Sensitivity to cisplatin induced apoptosis was also increased with loss of CLPTM1L expression by shRNA in H838 tumor cells (Figure 3B), measured by colorimetric Caspase 3/7 activity assay. Again resistance to cisplatin induced apoptosis was proportional to the amount of CLPTM1L transcript accumulation in the cells, with an r2 of 0.8847 (p = 1.3×10−4) between percent knockdown and relative caspase 3 activity (Figure S3B). The appearance of cells in culture is consistent with increased sensitivity to cisplatin induced apoptosis upon loss of CLPTM1L (Figure 3C).


Functional characterization of CLPTM1L as a lung cancer risk candidate gene in the 5p15.33 locus.

James MA, Wen W, Wang Y, Byers LA, Heymach JV, Coombes KR, Girard L, Minna J, You M - PLoS ONE (2012)

DNA damage induced apoptosis is regulated by CLPTM1L.A) Annexin V binding by flow cytometry of A549 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. B) Relative caspase 3/7 activity in H838 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. Error bars represent one standard deviation from the mean. **- p<0.01 * - p<0.02 by two-tailed Student’s T-Test. C) Micrographs of A549 cells with CLPTM1L knockdown after 24 hours treatment with 50µM cisplatin showing increased genotoxic cell death upon loss of CLPTM1L.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366984&req=5

pone-0036116-g003: DNA damage induced apoptosis is regulated by CLPTM1L.A) Annexin V binding by flow cytometry of A549 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. B) Relative caspase 3/7 activity in H838 cells with CLPTM1L knockdown after 48 hours treatment with cisplatin. Error bars represent one standard deviation from the mean. **- p<0.01 * - p<0.02 by two-tailed Student’s T-Test. C) Micrographs of A549 cells with CLPTM1L knockdown after 24 hours treatment with 50µM cisplatin showing increased genotoxic cell death upon loss of CLPTM1L.
Mentions: Annexin V immunofluorescence detected by flow cytometry was used as a marker of early apoptosis in A549 cells with or without CLPTM1L knockdown and cisplatin treatment. We observed a dose dependent increase in apoptosis with loss of CLPTM1L (Figure 3A). While only 16% of cells with vector alone were apoptotic after 10µM cisplatin treatment for 24 hours, 76% of cells with shCLPTM1L-3 were apoptotic. Resistance to cisplatin induced apoptosis was found to be proportional to the amount of CLPTM1L transcript accumulation in these cells, with an r2 of 0.9808 (p = 2×10−8) between percent knockdown and percent apoptotic cells (Figure S3A). Cisplatin concentrations at the lower limits of sensitivity were used to allow resolution of sensitivities conferred by different levels of CLPTM1L knockdown. The DNA damaging agents cisplatin and nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) both caused accumulation of DNA strand breaks independently of CLPTM1L expression as detected by the COMET method (Figure S4). Therefore, the observed increase in sensitivity tositivity to cisplatin upon loss of CLPTM1L is due to an effect on apoptosis or apoptotic signaling rather than an effect on levels of acute DNA damage. Sensitivity to cisplatin induced apoptosis was also increased with loss of CLPTM1L expression by shRNA in H838 tumor cells (Figure 3B), measured by colorimetric Caspase 3/7 activity assay. Again resistance to cisplatin induced apoptosis was proportional to the amount of CLPTM1L transcript accumulation in the cells, with an r2 of 0.8847 (p = 1.3×10−4) between percent knockdown and relative caspase 3 activity (Figure S3B). The appearance of cells in culture is consistent with increased sensitivity to cisplatin induced apoptosis upon loss of CLPTM1L (Figure 3C).

Bottom Line: This locus has been found by multiple genome wide association studies to be associated with lung cancer in both smokers and non-smokers.Bcl-xL accumulation was significantly decreased upon loss of CLPTM1L.Thus, this study implicates anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis and resistance to chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: MCW Cancer Center, Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
Cleft Lip and Palate Transmembrane Protein 1-Like (CLPTM1L), resides in a region of chromosome 5 for which copy number gain has been found to be the most frequent genetic event in the early stages of non-small cell lung cancer (NSCLC). This locus has been found by multiple genome wide association studies to be associated with lung cancer in both smokers and non-smokers. CLPTM1L has been identified as an overexpressed protein in human ovarian tumor cell lines that are resistant to cisplatin, which is the only insight thus far into the function of CLPTM1L. Here we find CLPTM1L expression to be increased in lung adenocarcinomas compared to matched normal lung tissues and in lung tumor cell lines by mechanisms not exclusive to copy number gain. Upon loss of CLPTM1L accumulation in lung tumor cells, cisplatin and camptothecin induced apoptosis were increased in direct proportion to the level of CLPTM1L knockdown. Bcl-xL accumulation was significantly decreased upon loss of CLPTM1L. Expression of exogenous Bcl-xL abolished sensitization to apoptotic killing with CLPTM1L knockdown. These results demonstrate that CLPTM1L, an overexpressed protein in lung tumor cells, protects from genotoxic stress induced apoptosis through regulation of Bcl-xL. Thus, this study implicates anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis and resistance to chemotherapy.

Show MeSH
Related in: MedlinePlus