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DNA damage repair is unaffected by mimicked heterozygous levels of BRCA2 in HT-29 cells.

Tannenbaum B, Mofunanya T, Schoenfeld AR - Int. J. Biol. Sci. (2007)

Bottom Line: However, the direct effects of BRCA2 heterozygosity remain unclear.Here, BRCA2 heterozygosity was mimicked in HT-29 colon cells by reducing levels of BRCA2 through stable RNA interference.Levels of p27 were also found to be slightly increased in cells with reduced BRCA2, perhaps contributing to the slower growth rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Adelphi University, Garden City, NY 11530, USA.

ABSTRACT
Functional loss of both alleles of the breast cancer susceptibility gene, BRCA2, facilitates tumorigenesis. However, the direct effects of BRCA2 heterozygosity remain unclear. Here, BRCA2 heterozygosity was mimicked in HT-29 colon cells by reducing levels of BRCA2 through stable RNA interference. No difference in RAD51 subcellular localization and focus formation was observed between control and mimicked heterozygous cell lines. DNA repair ability, as measured by colony survival following mitomycin C treatment and ultraviolet radiation exposure, was also unaffected by reduced levels of BRCA2. Interestingly, the growth rate of the mimicked BRCA2 heterozygous cell line was significantly lower than that of control cells. Increased expression of p53 in the mimicked heterozygous cells was observed, perhaps in response to BRCA2 deficiency. Levels of p27 were also found to be slightly increased in cells with reduced BRCA2, perhaps contributing to the slower growth rate. Overall, these results suggest that tumors are unlikely to arise directly from BRCA2 heterozygous cells without other genetic events such as loss of the wild-type BRCA2 allele and/or loss of p53 function or other cell cycle inhibitors.

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BRCA2 heterozygosity mimicked through RNA interference in HT-29 colon cells. (A) 25µg of lysates from parental, BRCA2 RNAi, and control RNAi cells were loaded on a SDS-10% polyacrylamide gel and a BRCA2 western blot was performed (top panel). Quantification of BRCA2 band intensities as a percent of the parental band is indicated. A non-specific band was utilized as a loading control in the experiment (middle panel). A lighter exposure of the non-specific band is shown (lower panel). (B) Western blot was performed as in (A), except lysates from parental HT-29 cells were compared to HT-29 cells stably infected with a shRNA construct targeting luciferase (Luc RNAi).
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Figure 1: BRCA2 heterozygosity mimicked through RNA interference in HT-29 colon cells. (A) 25µg of lysates from parental, BRCA2 RNAi, and control RNAi cells were loaded on a SDS-10% polyacrylamide gel and a BRCA2 western blot was performed (top panel). Quantification of BRCA2 band intensities as a percent of the parental band is indicated. A non-specific band was utilized as a loading control in the experiment (middle panel). A lighter exposure of the non-specific band is shown (lower panel). (B) Western blot was performed as in (A), except lysates from parental HT-29 cells were compared to HT-29 cells stably infected with a shRNA construct targeting luciferase (Luc RNAi).

Mentions: The heterozygous state of BRCA2 was mimicked in HT-29 colon cells via RNA interference in order to determine possible functional effects. BRCA2 RNAi cells were created via overnight incubation with 2 retroviruses containing shRNA BRCA2 targeting constructs (targeting BRCA2 bases 115-133 and 216-234) mixed together, as described in Materials and Methods. A control line of HT-29 cells was created with a control RNAi retrovirus, as also described. Following a 14 day selection, cells at fifty percent confluence were lysed and quantified for BRCA2 protein expression level by western blot to verify a reduction of BRCA2 protein levels to those likely to be seen with BRCA2 heterozygosity. In comparison to the parental and control RNAi cell lines, the BRCA2 RNAi cells expressed BRCA2 protein levels that were approximately half of the parental and control cells, verifying the mimic of BRCA2 heterozygosity (Figure 1a). The lower expression was not due to a non-specific effect of the shRNA since no reduction in BRCA2 was seen using a retroviral construct coding for a shRNA directed at the non-human target luciferase (Figure 1b).


DNA damage repair is unaffected by mimicked heterozygous levels of BRCA2 in HT-29 cells.

Tannenbaum B, Mofunanya T, Schoenfeld AR - Int. J. Biol. Sci. (2007)

BRCA2 heterozygosity mimicked through RNA interference in HT-29 colon cells. (A) 25µg of lysates from parental, BRCA2 RNAi, and control RNAi cells were loaded on a SDS-10% polyacrylamide gel and a BRCA2 western blot was performed (top panel). Quantification of BRCA2 band intensities as a percent of the parental band is indicated. A non-specific band was utilized as a loading control in the experiment (middle panel). A lighter exposure of the non-specific band is shown (lower panel). (B) Western blot was performed as in (A), except lysates from parental HT-29 cells were compared to HT-29 cells stably infected with a shRNA construct targeting luciferase (Luc RNAi).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2017108&req=5

Figure 1: BRCA2 heterozygosity mimicked through RNA interference in HT-29 colon cells. (A) 25µg of lysates from parental, BRCA2 RNAi, and control RNAi cells were loaded on a SDS-10% polyacrylamide gel and a BRCA2 western blot was performed (top panel). Quantification of BRCA2 band intensities as a percent of the parental band is indicated. A non-specific band was utilized as a loading control in the experiment (middle panel). A lighter exposure of the non-specific band is shown (lower panel). (B) Western blot was performed as in (A), except lysates from parental HT-29 cells were compared to HT-29 cells stably infected with a shRNA construct targeting luciferase (Luc RNAi).
Mentions: The heterozygous state of BRCA2 was mimicked in HT-29 colon cells via RNA interference in order to determine possible functional effects. BRCA2 RNAi cells were created via overnight incubation with 2 retroviruses containing shRNA BRCA2 targeting constructs (targeting BRCA2 bases 115-133 and 216-234) mixed together, as described in Materials and Methods. A control line of HT-29 cells was created with a control RNAi retrovirus, as also described. Following a 14 day selection, cells at fifty percent confluence were lysed and quantified for BRCA2 protein expression level by western blot to verify a reduction of BRCA2 protein levels to those likely to be seen with BRCA2 heterozygosity. In comparison to the parental and control RNAi cell lines, the BRCA2 RNAi cells expressed BRCA2 protein levels that were approximately half of the parental and control cells, verifying the mimic of BRCA2 heterozygosity (Figure 1a). The lower expression was not due to a non-specific effect of the shRNA since no reduction in BRCA2 was seen using a retroviral construct coding for a shRNA directed at the non-human target luciferase (Figure 1b).

Bottom Line: However, the direct effects of BRCA2 heterozygosity remain unclear.Here, BRCA2 heterozygosity was mimicked in HT-29 colon cells by reducing levels of BRCA2 through stable RNA interference.Levels of p27 were also found to be slightly increased in cells with reduced BRCA2, perhaps contributing to the slower growth rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Adelphi University, Garden City, NY 11530, USA.

ABSTRACT
Functional loss of both alleles of the breast cancer susceptibility gene, BRCA2, facilitates tumorigenesis. However, the direct effects of BRCA2 heterozygosity remain unclear. Here, BRCA2 heterozygosity was mimicked in HT-29 colon cells by reducing levels of BRCA2 through stable RNA interference. No difference in RAD51 subcellular localization and focus formation was observed between control and mimicked heterozygous cell lines. DNA repair ability, as measured by colony survival following mitomycin C treatment and ultraviolet radiation exposure, was also unaffected by reduced levels of BRCA2. Interestingly, the growth rate of the mimicked BRCA2 heterozygous cell line was significantly lower than that of control cells. Increased expression of p53 in the mimicked heterozygous cells was observed, perhaps in response to BRCA2 deficiency. Levels of p27 were also found to be slightly increased in cells with reduced BRCA2, perhaps contributing to the slower growth rate. Overall, these results suggest that tumors are unlikely to arise directly from BRCA2 heterozygous cells without other genetic events such as loss of the wild-type BRCA2 allele and/or loss of p53 function or other cell cycle inhibitors.

Show MeSH
Related in: MedlinePlus