p12DOC-1, a growth suppressor, associates with DNA polymerase alpha/primase

Abstract

p12(DOC-1) is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12(DOC-1) in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12(DOC-1) associates with DNA polymerase alpha/primase (pol-alpha:primase) in vitro and in cells. The pol-alpha:primase binding domain in p12(DOC-1) is mapped to the amino-terminal six amino acid (MSYKPN). The biological effect of p12(DOC-1) on pol-alpha:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12(DOC-1) suppresses DNA replication, leveling at approximately 50%. Similar results were obtained using the M13 single-stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12(DOC-1) affects the initiation step, not the elongation phase. The p12(DOC-1) suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol-alpha:primase or by its effect on cyclin-dependent kinase 2 (CDK2), a recently identified p12(DOC-1)-associated protein known to stimulate DNA replication by phosphorylating pol-alpha:primase. p12(DOC-1) suppresses CDK2-mediated phosphorylation of pol-alpha:primase. These data support a role of p12(DOC-1) as a regulator of DNA replication by direct inhibition of pol-alpha:primase or by negatively regulating the CDK2-mediated phosphorylation of pol-alpha:primase.

p12CDK2-AP1 Gene Therapy Strategy Inhibits Tumor Growth in an In vivo Mouse Model of Head and Neck Cancer

“…Cells reverted to a normal cellular morphology, lost the ability to grow in soft agar, and displayed an increase in cell doubling time (17), consistent with a potential role for p12 as a tumor suppressor gene in oral keratinocytes. Suppression of cell growth in a similar manner was also observed when malignant human keratinocytes were transfected with p12 (16). Additionally, p12 transfection into HCPC-1 cells significantly increased the fraction of apoptotic cells in p12-transfected as compared with null vector or untransfected controls (18).…”

“…The rodent p12 protein has 97% identity with that of humans (14). p12 is also implicated in S phase -associated growth suppression, through binding with DNA polymerase a/primase and/or CDK2 (15,16). Additionally, we have shown that the clinical behavior of p12 differs in normal and tumor oral mucosa, with reduced expression in 14.3% of oral dysplasias, and in 64% to 72% of HNSCC cases examined (12,13).…”

p12CDK2-AP1 Gene Therapy Strategy Inhibits Tumor Growth in an In vivo Mouse Model of Head and Neck Cancer

“…Cells reverted to a normal cellular morphology, lost the ability to grow in soft agar, and displayed an increase in cell doubling time (17), consistent with a potential role for p12 as a tumor suppressor gene in oral keratinocytes. Suppression of cell growth in a similar manner was also observed when malignant human keratinocytes were transfected with p12 (16). Additionally, p12 transfection into HCPC-1 cells significantly increased the fraction of apoptotic cells in p12-transfected as compared with null vector or untransfected controls (18).…”

“…The rodent p12 protein has 97% identity with that of humans (14). p12 is also implicated in S phase -associated growth suppression, through binding with DNA polymerase a/primase and/or CDK2 (15,16). Additionally, we have shown that the clinical behavior of p12 differs in normal and tumor oral mucosa, with reduced expression in 14.3% of oral dysplasias, and in 64% to 72% of HNSCC cases examined (12,13).…”

Knockdown of CDK2AP1 by RNA interference inhibits cell growth and tumorigenesis of human glioma

“……”

p12CDK2-AP1 interacts with CD82 to regulate the proliferation and survival of human oral squamous cell carcinoma cells

“…Increasing evidence indicates that p12 CDK2-AP1 interacts with other proteins such as DNA polymerase α/primase (9) and the homologous protein p14 (10). using a yeast two-hybrid system, our previous study also identified a novel unnamed protein product (uPP) that interacts with p12 CDK2-AP1 and inhibits the proliferation of 293T and HeLa cells (22).…”

p12CDK2-AP1 Gene Therapy Strategy Inhibits Tumor Growth in an In vivo Mouse Model of Head and Neck Cancer

“…Cells reverted to a normal cellular morphology, lost the ability to grow in soft agar, and displayed an increase in cell doubling time (17), consistent with a potential role for p12 as a tumor suppressor gene in oral keratinocytes. Suppression of cell growth in a similar manner was also observed when malignant human keratinocytes were transfected with p12 (16). Additionally, p12 transfection into HCPC-1 cells significantly increased the fraction of apoptotic cells in p12-transfected as compared with null vector or untransfected controls (18).…”

“…The rodent p12 protein has 97% identity with that of humans (14). p12 is also implicated in S phase -associated growth suppression, through binding with DNA polymerase a/primase and/or CDK2 (15,16). Additionally, we have shown that the clinical behavior of p12 differs in normal and tumor oral mucosa, with reduced expression in 14.3% of oral dysplasias, and in 64% to 72% of HNSCC cases examined (12,13).…”

Knockdown of CDK2AP1 by RNA interference inhibits cell growth and tumorigenesis of human glioma

“……”

p12CDK2-AP1 interacts with CD82 to regulate the proliferation and survival of human oral squamous cell carcinoma cells

“…Increasing evidence indicates that p12 CDK2-AP1 interacts with other proteins such as DNA polymerase α/primase (9) and the homologous protein p14 (10). using a yeast two-hybrid system, our previous study also identified a novel unnamed protein product (uPP) that interacts with p12 CDK2-AP1 and inhibits the proliferation of 293T and HeLa cells (22).…”

p12CDK2-AP1 Gene Therapy Strategy Inhibits Tumor Growth in an In vivo Mouse Model of Head and Neck Cancer

“…Cells reverted to a normal cellular morphology, lost the ability to grow in soft agar, and displayed an increase in cell doubling time (17), consistent with a potential role for p12 as a tumor suppressor gene in oral keratinocytes. Suppression of cell growth in a similar manner was also observed when malignant human keratinocytes were transfected with p12 (16). Additionally, p12 transfection into HCPC-1 cells significantly increased the fraction of apoptotic cells in p12-transfected as compared with null vector or untransfected controls (18).…”

“…The rodent p12 protein has 97% identity with that of humans (14). p12 is also implicated in S phase -associated growth suppression, through binding with DNA polymerase a/primase and/or CDK2 (15,16). Additionally, we have shown that the clinical behavior of p12 differs in normal and tumor oral mucosa, with reduced expression in 14.3% of oral dysplasias, and in 64% to 72% of HNSCC cases examined (12,13).…”

Knockdown of CDK2AP1 by RNA interference inhibits cell growth and tumorigenesis of human glioma

“……”

p12CDK2-AP1 interacts with CD82 to regulate the proliferation and survival of human oral squamous cell carcinoma cells

“…Increasing evidence indicates that p12 CDK2-AP1 interacts with other proteins such as DNA polymerase α/primase (9) and the homologous protein p14 (10). using a yeast two-hybrid system, our previous study also identified a novel unnamed protein product (uPP) that interacts with p12 CDK2-AP1 and inhibits the proliferation of 293T and HeLa cells (22).…”