Expression of DNA polymerase β cancer-associated variants in mouse cells results in cellular transformation

Abstract

Thirty percent of the 189 tumors studied to date express DNA polymerase ␤ variants. One of these variants was identified in a prostate carcinoma and is altered from isoleucine to methionine at position 260, within the hydrophobic hinge region of the protein.Another variant was identified in a colon carcinoma and is altered at position 289 from lysine to methionine, within helix N of the protein. We have shown that the types of mutations induced by these cancer-associated variants are different from those induced by the wild-type enzyme. In this study, we show that expression of the I260M and K289M cancer-associated variants in mouse C127 cells results in a transformed phenotype in the great majority of cell clones tested, as assessed by focus formation and anchorageindependent growth. Strikingly, cellular transformation occurs after a variable number of passages in culture but, once established, does not require continuous expression of the polymerase ␤ variant proteins, implying that it has a mutational basis. Because DNA polymerase ␤ functions in base excision repair, our results suggest that mutations that arise during this process can lead to the onset or progression of cancer.base excision repair ͉ DNA repair ͉ mutagenesis S pontaneous DNA damage occurs at a rate of Ϸ10,000 lesions per cell per day, and much of this damage is repaired by the base excision repair (BER) machinery (1, 2). The BER system plays a critical role in maintaining cellular genomic stability. During BER, damaged bases are removed by a DNA glycosylase, followed by incision of the DNA by AP endonuclease (APE) at a position that is usually 5Ј to the lesion, leaving a nick with a 3ЈOH and a 5Ј deoxyribose phosphate (2). DNA polymerase beta (pol ␤) binds to the nick, removes the deoxyribose phosphate with its DRP lyase activity (3), and fills in the single nucleotide gap, using its DNA polymerase activity (4).Fifty-eight of the 189 tumors characterized to date express DNA pol ␤ variant proteins (for review, see ref. 5) (6). Of these, 28 (48%) expressed variants with single amino acid alterations, seven expressed truncated forms of pol ␤, and eight expressed multiple variant forms of pol ␤. These mutations are absent from normal tissue from the same individuals and are not among the common polymorphisms found within the pol ␤ gene (http:͞͞ egp.gs.washington.edu͞data͞polb) (5, 7). In addition, an alternative splice variant of pol ␤ in which exon 11 is deleted was expressed in 15 tumors. This splice variant appears to interfere with BER (8). This exon 11 splice variant has been detected in normal tissue, including normal tissue isolated from 2 of 15 patients with tumors, and its link to cancer etiology remains controversial (9-12). Each of the tumors characterized to date also contain the wild-type (WT) pol ␤ allele.The I260M variant of pol ␤ was identified in a prostate carcinoma (13). Isoleucine 260 is located within a hydrophobic hinge region that appears to function in the movement of the fingers subdomain upon interaction of the polymeras...

Human POLB Gene Is Mutated in High Percentage of Colorectal Tumors

“…Variant sequences were confirmed by direct sequencing at the Keck DNA Sequencing Facility, Yale University School of Medicine. For retroviral infection into mouse cells, the wild-type pol ␤ cDNA with a C-terminal hemagglutinin (HA) tag was cloned into the pRVYtet retroviral vector as described previously (23). pol ␤ variant sequences were generated and confirmed using the above protocol.…”

“…We have shown that K289M variant protein induces cellular transformation in immortalized mouse epithelial cells by increasing the mutation frequency of the cells (22,23). We have also shown that this variant is a sequence context-dependent mutator, in that it induces mutations at a frequency 16 times higher than wild-type (WT) pol ␤ within a nucleotide sequence context found in the adenomatous polyposis coli gene that is frequently mutated in colon cancer (22,24).…”

Estrogen withdrawal, increased breast cancer risk and the KRAS-variant

“…37 Anchorage independent growth was assessed as described previously. 38 After thawing and growing cells until confluence in EGF supplemented media (20 ng/ml), cells were plated into conditions of study for 2 passages. For estrogen depletion experiments phenol red free DMEM/F12 medium (Invitrogen) and 5% charcoal-stripped horse serum (Thermo Fisher), were used, and Tamoxifen or estrogen was added to a final concentration of 1uM after the first passage, as appropriate.…”

Is Base Excision Repair a Tumor Suppressor Mechanism?

“…[11][12][13] Specifically, expression of either the K289M colon or I260M prostate cancer Pol β variant in mouse fibroblasts, in the presence of endogenous wild-type Pol β, results in cellular transformation as assessed by focus formation and anchorage independent growth. [11][12][13] A mutagenic basis for the transforming activity of the Pol β variants is suggested by the findings that different clones of cells expressing these polymerases were not immediately transformed but acquired the transformed phenotype after a variable number of passages and that the transformed phenotype persisted even after the expression of the Pol β variant was extinguished. The latter finding indicates that the Pol β variants induce transformation by a "hit-and-run" mechanism in which the continued expression of the initiating event is not required to maintain the transformed state.…”