Joanna Groden

RecQ Family Members Combine Strand Pairing and Unwinding Activities to Catalyze Strand Exchange

RecQ helicases are critical for maintaining genomic integrity. In this study, we show that three RecQ members (WRN, deficient in the Werner syndrome; BLM, deficient in the Bloom syndrome; and Drosophila melanogaster RecQ5b (dmRecQ5b)) possess a novel strand pairing activity. Furthermore, each of these enzymes combines this strand pairing activity with its inherent DNA unwinding capability to perform coordinated strand exchange. In this regard, WRN and BLM are considerably more efficient than dmRecQ5b, apparently because dmRecQ5b lacks conserved sequences C-terminal to the helicase domain that contribute to DNA binding, strand pairing, and strand exchange. Based on our findings, we postulate that certain RecQ helicases are structurally designed to accomplish strand exchange on complex replication and recombination intermediates. This is highly consistent with proposed roles for RecQ members in DNA metabolism and the illegitimate recombination and cancer-prone phenotypes associated with RecQ defects.

β-Catenin/Wnt Signaling Regulates Expression of the Membrane Type 3 Matrix Metalloproteinase in Gastric Cancer

Activation of Wnt signaling through beta-catenin dysregulation occurs in numerous human tumors, including gastric cancer. The specific consequences of Wnt signaling in gastric cancer, however, are not well characterized. This study shows that the introduction of mutant beta-catenin into gastric cancer cell lines by adenoviral infection enhances invasiveness and proliferation and up-regulates the expression of the gene encoding the matrix metalloproteinase (MMP) family member membrane type 3 MMP (MT3-MMP). Up-regulation of MT3-MMP is critical to the invasive phenotype as shown by small interfering RNA (siRNA) studies. Immunohistochemical staining also showed that MT3-MMP was highly expressed in gastric cancers with activating beta-catenin mutations. These observations suggest that Wnt activation may contribute to gastric cancer progression by increasing the invasiveness of neoplastic cells in the stomach via up-regulation of MT3-MMP expression.

PHOSPHORYLATION OF THE TUMOR SUPPRESSOR ADENOMATOUS POLYPOSIS COLI (APC) BY THE CYCLIN-DEPENDENT KINASE P34CDC2

Mutations in the tumor suppressor geneAPC invariably lead to the development of colorectal cancer. The vast majority of these mutations are nonsense or frameshifts resulting in nonfunctional, truncated APC protein products. Eleven cyclin-dependent kinase (CDK) consensus phosphorylation sites have been identified in the frequently deleted carboxyl-terminal region of APC; loss of these phosphorylation sites by mutation could therefore compromise the ability of APC to inhibit cell growth. This report demonstrates that immunoprecipitates of full-length, but not truncated, APC protein include a mitosis-specific kinase activity in vivo. Biochemical and Western analysis of these immunoprecipitates confirms the presence of the CDK p34 cdc2 . We also show that APC is a substrate for recombinant human p34 cdc2 -cyclin B1. Modification of APC by p34 cdc2 implicates phosphorylation as a mechanism for regulating APC function via a link to the cell cycle.

Dysregulation of β-Catenin Expression Correlates With Tumor Differentiation in Pancreatic Duct Adenocarcinoma

Background: β-Catenin functions as an integral part of the E-cadherin/catenin adhesion complex to maintain epithelial cell integrity. β-Catenin also functions as part of the Wnt signal transduction pathway to transmit growth-promoting signals to the nucleus via its interactions with Tcf/Lef transcription factors. Previous reports have demonstrated altered β-catenin expression in numerous tumor types; however, reports regarding β-catenin expression in pancreatic cancer have been conflicting.Methods: β-Catenin expression was examined in 10 pancreatic cancer cell lines by Western and Northern analysis and by immunofluorescence. Expression was also examined by immunohistochemistry in 57 primary pancreatic cancers and 7 foci of carcinoma-in-situ.Results: Reduced expression of β-catenin was observed in 4 of 10 pancreatic cancer cell lines. Reduced membranous expression was noted in 32 pancreatic cancers (56%) and correlated with loss of tumor differentiation. Nuclear β-catenin expression was identified in two tumors (4%). β-Catenin expression was present in all seven foci of carcinoma-in-situ; however, nuclear expression was predominant in four of the seven cases.Conclusions: Alterations in β-catenin expression are common in pancreatic cancer; however, signaling and adhesion functions may be perturbed at different times during tumor progression. Therefore, dysregulation of β-catenin may contribute to the development and progression of this disease through distinct mechanisms.

Interaction between two isoforms of the NF2 tumor suppressor protein, merlin, and between merlin and ezrin, suggests modulation of ERM proteins by merlin

The product of the neurofibromatosis type II (NF2) tumor suppressor gene, merlin, is closely related to the ezrin‐radixin‐moesin (ERM) family, a group of proteins believed to link the cytoskeleton to the plasma membrane. Mutation in the NF2 locus is associated with Schwann cell tumors (schwannomas). The two predominant merlin isoforms, I and II, differ only in the carboxy‐terminal 16 residues and only isoform I is anti‐proliferative. Merlin lacks an actin‐binding domain conserved among ezrin, radixin and moesin. Because merlin, ezrin and moesin are co‐expressed in Schwann cells, and all homodimerize, we have examined whether merlin and ezrin dimerize with one another. We found by immunoprecipitation and yeast two‐hybrid assays that both merlin isoforms interact with ezrin. The interaction occurs in a head‐to‐tail orientation, with the amino‐terminal half of one protein interacting with the carboxy‐terminal half of the other. The two merlin isoforms behave differently in their interaction with ezrin. Isoform I binds only ezrin whose carboxy‐terminus is exposed, whereas isoform II binds ezrin regardless of whether ezrin is in the open or closed conformation. The heterodimerization of merlin is a much stronger interaction than the interaction between either merlin isoform and ezrin, and can inhibit merlin‐ezrin binding. This suggests that, in vivo, merlin dimerization could regulate merlin‐ERM protein interaction, and could thus indirectly regulate other interactions involving ERM proteins. J. Neurosci. Res. 62:491–502, 2000.

Novel germline APC variants in patients with multiple adenomas

Chain‐terminating germline APC mutations are responsible for adenomatous polyposis coli (APC). In the present work, we tested the hypothesis that germline APC mutations may be present in some patients with a milder phenotype, i.e., multiple synchronous colorectal adenomas. Eighteen patients with 3 or more colorectal adenomas at endoscopy (within a 6‐month period) were ascertained from a series of subjects undergoing endoscopic examination. Their blood DNAs were analysed for the presence of germline mutations in the APC coding region by single‐strand polymorphism analysis. Ten unrelated polyp‐free subjects and 101 unrelated APC patients were used as controls in the molecular analyses. Five of the eighteen patients carried novel germline APC variants or rare polymorphisms. These were various in site (from the splice acceptor site of intron 7 to the end of exon 15) and type (splice‐site, missense, and chain‐terminating mutations). Only one of ten polyp‐free individuals carried a silent APC variant and none of these variants was found in the 101 APC controls. A first‐ or second‐degree family history of colorectal cancer was reported by 4 of the 5 patients carrying a germline APC variant. In conclusion, novel APC germline variants were detected in patients with multiple synchronous adenomas. This suggests that the development of sporadic adenomas, in some instances, is associated with the presence of minor germline variants of the APC gene and that the spectrum of germline APC functional mutations may be larger than previously thought. Genes Chromosomes Cancer 22:257–267, 1998.

The APC Tumor Suppressor Promotes Transcription- Independent Apoptosis In vitro

The APC tumor suppressor is found in nonproliferating epithelial cells of the colonic crypts and is mutated in most colorectal tumors. To understand the function of APC in normal epithelium and how its loss leads to tumor formation, we tested whether APC is a mediator of apoptosis using an in vitro assay that monitors caspase-3-mediated cleavage of lamin B protein or a colorimetric substrate in a cell-free Xenopus egg extract. Recombinant APC protein accelerates apoptosis-associated caspase activity independently of ongoing transcription and protein synthesis. Conversely, the addition of mutant APC and immunodepletion of Xenopus APC decelerates apoptosis-associated caspase activity. Acceleration of apoptosis by APC is abolished by the caspase-8 inhibitor Z-IETD-FMK, demonstrating that caspase-8 is an essential component of APC-mediated apoptosis. These results suggest that the induction of apoptosis may be one role of APC in tumor suppression and that this mechanism is independent of β-catenin-mediated effects on transcription.

Regenerative lesions in ulcerative colitis are characterized by microsatellite mutation

An increased risk of colon cancer has been observed in individuals with long‐standing ulcerative colitis (UC). In order to identify molecular genetic markers for the development of neoplasia in UC individuals, we isolated DNA from normal, regenerative, and dysplastic mucosa, as well as from colon carcinomas from UC patients, and evaluated it for the presence of mutations in microsatellite DNA sequences. DNAs isolated from regenerative mucosa displayed microsatellite mutation. These observations suggest that DNA mutation is an early event in the UC disease process. Genes Chromosom. Cancer 19:170–175, 1997.

Novel protein isoforms of the APC tumor suppressor in neural tissue

The conventional protein isoform of the APC tumor suppressor is 310 kD and is encoded by exons 1 – 15 of the APC gene. Other RNAs are expressed from the APC gene and include one form that contains an exon upstream of exon 1, designated BS, but this transcript does not include exon 1. This transcript recently has been shown to be enriched in non-dividing, terminally-differentiated cells (Santoro and Groden, 1997). To determine if the BS-containing transcript encoded an alternate APC protein isoform, we generated and affinity-purified a polyclonal antibody directed to protein sequence predicted by exon BS. The BS antibody labeled a band of ∼300 kD on immunoblots of cerebral and cerebellar tissue from adult human, baboon, rat and mouse. These same tissue lysates also contained prominent BS-reactive proteins of 290 kD, 200 kD and 150 kD. Lysates from mitotically active cells did not contain these APC isoforms. To verify that BS-reactive proteins were APC isoforms, BS-immunoprecipitates were blotted and labeled with commercially available APC antibodies. All four high molecular weight BS-antibody-precipitated proteins were recognized by antibodies directed against epitopes encoded by APC exons 2 and 15. BS isoforms were not, however, labeled with antibodies to an epitope encoded by APC exon 1, consistent with the prediction that BS – APC isoforms lack the domain encoded by these sequences. Like conventional APC, at least one of the four BS – APC protein isoforms also interacts with β-catenin. BS – APC isoforms that lack exon 1-encoded sequences are incapable of dimerization with the conventional form of APC, yet retain the ability to bind β-catenin. Such isoforms are likely to be functionally distinct from the conventional APC protein.

Chain‐terminating mutations in the APC gene lead to alterations in APC RNA and protein concentration

APC mutations introduce premature stop codons into the open reading frame of the gene, leading to the formation of truncated tumor suppressor proteins. Both RNA and protein levels are likely to be profoundly altered by such nonsense mutations. To test this hypothesis, Western blotting and RT‐PCR strategies were used to characterize mutant and normal APC protein and APC RNA concentrations in lymphoblastoid cell lines from 22 unrelated polyposis patients carrying different APC mutations. Variable levels of truncated APC peptides were observed in 14 of 14 cell lines with APC mutations within exon 15. No truncated APC protein was detected in six of eight cell lines with APC mutations located 5′ of exon 15. Mutations located in exon 15 showed mutant RNA underrepresentation in four of eight cell lines, whereas mutations located 5′ of exon 15 showed RNA reduction in five of six cell lines. These findings indicate that a two‐ to threefold decrease in RNA concentration is common when APC alleles carry chain‐terminating mutations. They also suggest that the severe decrease of truncated APC protein observed in some cell lines is due to mechanisms acting at the protein level. Genes Chromosomes Cancer 22:278–286, 1998.