Cells transduce mechanical forces into biochemical signals; traditionally these processes are thought to occur through direct effects on the cell membrane, the cytoskeleton, or specific transmembrane proteins. In multicellular tissues mechanical forces alter intercellular spacing through redistribution of interstitial fluid. Recent morphological and biochemical observations, bolstered by analytical modeling, support a new paradigm for mechanotransduction arising from constitutive growth factor shedding into a dynamically regulated interstitial volume.

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...

Previous investigations have shown that Ϸ35% of the 90 tumors analyzed to date contain mutations within the DNA polymerase ␤ (pol ␤) gene. The existence of pol ␤ mutations in a substantial fraction of human tumors studied suggests a link between DNA pol ␤ and cancer. A DNA pol ␤ variant, in which Lys-289 has been altered to Met, was identified previously in a colorectal carcinoma. The K289M protein was expressed in mouse L cells containing the cII mutational target. The DNA was packaged and used to infect bacterial cells to obtain the spontaneous mutation frequency. We found that expression of K289M in the mouse cells resulted in a 2.5-fold increase in the mutation frequency. What was most interesting was that expression of K289M in these cells resulted in a 16-fold increase in the frequency of C to G or G to C base substitutions at a specific site within the cII target. By using this cII target sequence, kinetic analysis of the purified K289M protein revealed that it was able to misincorporate dCTP opposite template C and dGTP opposite template G with significantly higher efficiency than the wild-type pol ␤ protein. We provide evidence that misincorporation of nucleotides by K289M results from altered positioning of the DNA within the active site of the enzyme. Our data are consistent with the interpretation that misincorporation of nucleotides resulting from altered DNA positioning by the K289M protein has the potential to result in tumorigenesis or neoplastic progression.M utations in the gene encoding DNA polymerase ␤ (pol ␤) have been identified in human colorectal, prostate, lung, and breast carcinomas and mouse lymphomas (1-5). Thus far, only 90 tumors have been analyzed for mutations within the pol ␤ coding sequence, and mutations are present in 35% of these tumors. The pol ␤ tumor-associated mutations are found only in the tumor, and not in normal tissue from the same patient, implying that they represent sporadic mutations underlying neoplastic disease. Furthermore, the mutations identified in these tumors are not present in the pol ␤ gene of 124 normal individuals (6). Also of interest is that pol ␤ is located within the proximal region of the short arm of chromosome 8 (p12-p11), a region that is frequently lost in a variety of human tumors, including colorectal and prostate carcinomas (7). These studies suggest a link between mutations within the pol ␤ gene and carcinogenesis. Another piece of evidence that is consistent with a role for pol ␤ in cancer is its interaction with the tumor suppressor protein p53 (8, 9). The p53 protein stabilizes pol ␤ at an abasic site. An alteration of the p53-pol ␤ interaction could result in less efficient DNA repair, which may contribute to the development of neoplastic disease. Most interestingly, a pol ␤ mutant with an 87-bp deletion, which has been found in primary colorectal, lung, and breast adenocarcinomas (3,5,10), is dominant to the WT enzyme and disrupts its base excision repair (BER) activity if expressed in human cell lines (11,12). It is quite possible t...

Purpose: To evaluate the potential of targeted photoacoustic imaging as a noninvasive method for detection of follicular thyroid carcinoma.Experimental Design: We determined the presence and activity of two members of matrix metalloproteinase family (MMP), MMP-2 and MMP-9, suggested as biomarkers for malignant thyroid lesions, in FTC133 thyroid tumors subcutaneously implanted in nude mice. The imaging agent used to visualize tumors was MMP-activatable photoacoustic probe, Alexa750-CXeeeeXPLGLAGrrrrrXK-BHQ3. Cleavage of the MMP-activatable agent was imaged after intratumoral and intravenous injections in living mice optically, observing the increase in Alexa750 fluorescence, and photoacoustically, using a dual-wavelength imaging method.Results: Active forms of both MMP-2 and MMP-9 enzymes were found in FTC133 tumor homogenates, with MMP-9 detected in greater amounts. The molecular imaging agent was determined to be activated by both enzymes in vitro, with MMP-9 being more efficient in this regard. Both optical and photoacoustic imaging showed significantly higher signal in tumors of mice injected with the active agent than in tumors injected with the control, nonactivatable, agent.Conclusions: With the combination of high spatial resolution and signal specificity, targeted photoacoustic imaging holds great promise as a noninvasive method for early diagnosis of follicular thyroid carcinomas.

Studies show that 30% of 189 tumors sequenced to date express variants of the polymerase beta (pol beta) protein that are not present in normal tissue. This raises the possibility that variants of pol beta might be linked to the etiology of cancer. Here, we characterize the I260M prostate-cancer-associated variant of pol beta. Ile260 is a key residue of the hydrophobic hinge that is important for the closing of the polymerase. In this study, we demonstrate that the I260M variant is a sequence context-dependent mutator polymerase. Specifically, I260M is a mutator for misalignment-mediated errors in dipyrimidine sequences. I260M is also a low-fidelity polymerase with regard to the induction of transversions within specific sequence contexts. Our results suggest that the hinge influences the geometry of the DNA within the polymerase active site that is important for accurate DNA synthesis. Importantly, characterization of the I260M variant shows that it has a functional phenotype that could be linked to the etiology or malignant progression of human cancer.