Deleted in oral cancer-1 expression upregulates proapoptosis elements in microsatellite-unstable human colorectal cancer.
BACKGROUND
We previously reported differential expression of the growth suppressor, deleted in oral cancer-1 (DOC-1), in microsatellite-unstable (MSI+) versus microsatellite-stable colorectal cancer (CRC) cell lines. MSI+ CRC cell lines demonstrated decreased DOC-1 expression and decreased apoptosis. Transfection of wild-type DOC-1 into an MSI+ cell line (SW48) resulted in increased apoptosis. We undertook our current experiment to identify specific elements modulated by DOC-1 expression that result in increased apoptosis.


METHODS
SW48 is an MSI+ CRC cell line that does not constitutively express DOC-1. SW48 was suspended in culture medium and incubated to 60% confluence. Half the plates were transfected with cytomegalovirus (CMV)-DOC-1. At 30 hours, RNA and protein were isolated with Trizol. Complementary DNA microarray was performed to compare SW48(CMV-DOC-1) with SW48, which lacks DOC-1. Signal intensity was analyzed by GenePix Pro 3.0 software. Expression ratios </=.67 and >/ 1.5 were considered significant. Poor-quality spots were flagged and excluded from analysis. Real-time polymerase chain reaction was performed to determine DOC-1 levels in both cell lines.


RESULTS
Successful transfection of DOC-1 was confirmed by real-time polymerase chain reaction and by Western blot. Microarray revealed significant differential expression of DOC-1, as expected. Increased DOC-1 expression in SW48(CMV-DOC-1) was associated with significantly increased expression of proapoptosis components of the caspase cascade (CASP7, CASP9) and bcl2/bax pathway (BNIP3, BNIP3L, BID).


CONCLUSIONS
DOC-1 expression promotes apoptosis by upregulation of specific elements of the caspase cascade and bcl2/bax pathways. DOC-1 therefore deserves further study as a candidate for the therapeutic modulation of apoptosis in MSI+ CRC.
Sotsky Kent
Tara
T
Departments of Surgery, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, New York, USA.
Yuan
Ziqiang
Z
Miller
Agnes
A
Weber
Thomas K
TK
D017209:Apoptosis / Q000235:genetics*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D015854:Up-Regulation
C096151:CDK2AP1 protein, human; D025521:Tumor Suppressor Proteins