Biomedical subjects

D L Rimm

Publications and source records attributed to D L Rimm.

At least 37 records · Page 2Linked to original sources

PubMed1999

Frequent nuclear/cytoplasmic localization of beta-catenin without exon 3 mutations in malignant melanoma.

Beta-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3beta phosphorylation sites near the beta-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render beta-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3beta, adenomatous polyposis coli, and axin proteins. As a result, beta-catenin accumulates in the cytosol and nucleus and activates T-cell factor/ lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have beta-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790-1792). To assess the role of beta-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of beta-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in beta-catenin was found in only one case (codon 45 Ser-->Pro). Our findings demonstrate that beta-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of beta-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor.

Cell Nucleus↗

PubMed1999

Controversies at the cytoplasmic face of the cadherin-based adhesion complex.

Cadherin-mediated cell-cell interactions are modulated by protein interactions at the cytoplasmic face of the membrane. Recent work has shown that phosphorylation of both p120(ctn) and beta-catenin affects their interaction with cadherins and the molecular connections to the cytoskeleton. The cytoskeletal connections most probably include interactions between alpha-catenin, and/or alpha-actinin, vinculin, ZO-1, actin and possibly spectrin.

Animals↗

PubMed1999

PECAM-1 (CD31) functions as a reservoir for and a modulator of tyrosine-phosphorylated beta-catenin.

Catenins function as regulators of cellular signaling events in addition to their previously documented roles in adherens junction formation and function. Evidence to date suggests that beta and gamma catenins can act as signaling molecules, bind transcriptional factors and translocate to the nucleus. Beta- and gamma-catenin are also major substrates for protein tyrosine kinases, and tyrosine phosphorylation of junctional proteins is correlated with decreased adhesiveness. One way in which catenin functions are modulated is by dynamic incorporation into junctional complexes which controls, in part, the cytoplasmic levels of catenins. Here we show that: (1) vascular endothelial growth factor (VEGF) induces beta-catenin tyrosine phosphorylation in a time-, and dose-dependent manner and that VEGF receptors co-localize to areas of endothelial cell-cell contact in vitro and in vivo. (2) Platelet-endothelial cell adhesion molecule (PECAM)-1 can function as a reservoir for, and modulator of, tyrosine phosphorylated beta-catenin. (3) PECAM-1 can prevent beta-catenin nuclear translocation in transfected SW480 colon carcinoma cells. We suggest that PECAM-1 may play a role in modulating beta-catenin tyrosine phosphorylation levels, localization and signaling and by doing so, functions as an important modulator of the endothelium.

Animals↗

PubMed1999

Beta- and gamma-catenin mutations, but not E-cadherin inactivation, underlie T-cell factor/lymphoid enhancer factor transcriptional deregulation in gastric and pancreatic cancer.

Adenomatous polyposis coli (APC) mutations are present in >70% of colon cancers. The APC protein binds to beta-catenin (beta-cat), a protein first identified because of its role in E-cadherin (E-cad) cell adhesion. In some colon cancers lacking APC defects, mutations in presumptive glycogen synthase kinase 3beta phosphorylation sites near the beta-cat NH2 terminus appear to render beta-cat resistant to regulation by APC and glycogen synthase kinase 3beta. In cells with APC or beta-cat defects, beta-cat is stabilized and, in turn, binds to and activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef) transcription factors. To further explore the role of APC, beta-cat, Tcf, and E-cad defects in gastrointestinal cancers, we assessed gastric and pancreatic cancers for constitutive Tcf transcriptional activity (CTTA). Two of four gastric and two of eight pancreatic cancer lines showed CTTA. One gastric and one pancreatic cancer had mutations in the NH2-terminal phosphorylation sites of beta-cat. The other gastric cancer with CTTA had a missense mutation at serine 28 of gamma-cat, a potential phosphorylation site in this beta-cat-related protein. Although E-cad is an important binding partner for beta-cat and gamma-cat, E-cad inactivation did not result in CTTA. The beta-cat and gamma-cat mutant proteins identified in our studies strongly activated Tcf transcription in vitro, whereas beta-cat mutant proteins with large NH2-terminal deletions had only modest effects on Tcf. Our results suggest a role for Tcf deregulation in gastric and pancreatic cancer, resulting from beta-cat and gamma-cat mutations in some cases and, in others, from yet to be defined defects. Furthermore, these data imply that the consequences of APC and beta-cat mutations are distinct from the effects of E-cad inactivation.

Adenomatous Polyposis Coli Protein↗

PubMed1998

Loss of p120ctn in human colorectal cancer predicts metastasis and poor survival.

The p120ctn protein (formerly p120CAS) is an armadillo family member that associates directly with the cytoplasmic tail of E-cadherin and participates in the junctional complex responsible for cell-cell adhesion. Since reduced cell-cell adhesion is associated with metastasis in colorectal cancer and other neoplasms, we hypothesize that reduced expression of p120ctn may be related to metastasis in colorectal tumors. Here we describe a study of p120ctn expression in 44 primary human colorectal adenocarcinomas. As detected by immunohistochemical methods, we find altered p120ctn staining patterns in 86% of the cases. Regional complete loss of expression is seen in 18% of the cases, and it correlates with high stage disease and nodal metastasis as well as decreased survival. Although this is a preliminary study, it suggests that downregulation of p120ctn in colon cancer may be associated with metastasis and poor clinical outcome.

Adult↗

PubMed1998

Polymerase chain reaction-based detection of clonality as a non-morphologic diagnostic tool for fine-needle aspiration of the breast.

BACKGROUND: Fine-needle aspiration (FNA) of breast specimens can be difficult and between 10-25% of the lesions ultimately are classified as "atypical," even by the most experienced cytopathologist. The goal of this study was to identify a molecular mechanism that reliably distinguishes benign and malignant (or pre-malignant) lesions and that could be used as an adjunct in these morphologically ambiguous cases. METHODS: Because all malignancies represent clonal proliferations, assessment of clonality represents a potential molecular mechanism for making this distinction. Excess material preserved from breast FNAs was examined using the human androgen receptor locus clonality assay. This assay allows determination of clonality on the basis of X chromosome inactivation as detected by polymerase chain reaction analysis of genomic DNA after methylase-sensitive restriction digestion. RESULTS: In this pilot study, 25 cases showed reproducible results. All malignant cases (9 of 9) were monoclonal, whereas 10 of 12 benign cases were polyclonal. Of four atypical cases, two were monoclonal and both were found to be malignant after surgical resection. Monoclonality was observed in two benign cases that were hyperplastic lesions. CONCLUSIONS: These preliminary results suggest that this test may provide a non-morphologic molecular mechanism for the objective categorization of breast FNAs.

Adult↗

PubMed1998

Expression of c-met is a strong independent prognostic factor in breast carcinoma.

BACKGROUND: The c-met protooncogene encodes the met protein, the receptor for scatter factor/hepatocyte growth factor, a growth factor that modulates the motility and stable interaction of the epithelial cells. This study assesses the expression of met receptor in breast carcinoma and its prognostic value with respect to survival. METHODS: Immunofluorescence was used to evaluate 91 archival breast carcinoma specimens using a polyclonal antibody to the cytoplasmic domain of the receptor. Cases were scored by two pathologists on a percentage basis and then converted to binary scores (positive or negative) on the basis of a bimodal distribution. RESULTS: Strong expression of met was found in 20 invasive ductal breast tumor specimens (22%). The 5-year survival of patients whose tumors showed decreased met expression was 89%, in contrast to a 52% 5-year survival rate in patients whose tumors expressed met (P = 0.008). This trend also was observed in patients without lymph node metastases at presentation, in whom met negative patients had a 95% 5-year survival compared with only 62% for met positive patients (P = 0.006) Multivariate analysis using the Cox proportional hazards model showed met expression to be an independent predictor of survival, with a predictive value nearly equivalent to that associated with lymph node status. CONCLUSIONS: The authors conclude that expression of met in patients with invasive ductal carcinoma of the breast is a strong, independent predictor of decreased survival and may be a useful prognostic marker with which to identify a subset of patients with more aggressive disease.

Adult↗

PubMed1998

Dynamic interaction of PTPmu with multiple cadherins in vivo.

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPmu associates with the cadherin-catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S. M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977- 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPmu interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPmu and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPmu and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPmu from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPmu in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513-1517) have asserted that the association we observed between PTPmu and the cadherin-catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPmu, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPmu obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPmu antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPmu and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.

Animals↗

PubMed1998

A mutation in alpha-catenin disrupts adhesion in clone A cells without perturbing its actin and beta-catenin binding activity.

Cadherin mediated cell-cell adhesion requires cytoplasmic connections to the cytoskeleton mediated by alpha-catenin. Original descriptions of the catenins, as well as our own in vitro studies, have suggested that this connection was mediated by the interaction of alpha-catenin to actin. Loss of adhesion in the human colon carcinoma cell line "Clone A" is the result of an internal deletion mutation of 158 residues near the N-terminus of the protein resulting in an 80 kD mutated protein. Transfection of these cells with the full length protein restores the normal adhesive phenotype. We have characterized this mutant protein in efforts to understand the normal function of alpha-catenin and, in particular, the region deleted in the Clone A mutant. Co-precipitation experiments using whole cell lysates indicate that the mutant form of alpha-catenin binds beta-catenin and plakoglobin, and can form a structural complex with E-cadherin via these interactions. Actin co-sedimentation assays show that the recombinant mutant binds and bundles F-actin and binds both actin and beta-catenin simultaneously, as seen with wild type alpha-catenin. These results suggest that the stabilization of the E-cadherin-catenin complex may be mediated by factors beyond its direct interaction with actin. We conclude that a region near the N-terminus of alpha-catenin mediates additional interactions between the adhesive complex and the cytoskeleton that are critical for functional adhesion.

Actins↗

PubMed1998

The expression of p120ctn protein in breast cancer is independent of alpha- and beta-catenin and E-cadherin.

Several studies have reported loss or alteration of expression of E-cadherin in breast cancer and more recently changes in levels of expression of the catenins. We used immunofluorescence to examine E-cadherin, alpha-catenin, beta-catenin, and p120ctn (formerly p120CAS) expression in 91 cases of invasive ductal carcinoma. As expected, all four proteins co-localize to the junctional regions of the cells. Although nuclear localization has been described for beta-catenin in colonic polyps, no examples were found in these breast cancer cases. We found that, although alteration is common in the catenins and E-cadherin, complete loss, as exemplified by E-cadherin in lobular carcinoma (where E-cadherin is frequently mutated), is rarely seen. In contrast, the catenin-related protein p120ctn shows an expression pattern that is significantly unrelated to the other catenins (or E-cadherin), including complete loss of expression in approximately 10% of the cases. No statistically significant correlations with traditional prognostic indicators were observed with any of these proteins. We conclude 1) that expression of E-cadherin and alpha- and beta-catenin are generally retained at the membrane although frequently reduced or altered, 2) that complete loss of p120ctn expression is seen in approximately 10% of the cases, and 3) that there is a significant correlation in the expression of E-cadherin and the catenins but no correlation between these molecules and p120ctn, suggesting an absence of coordinate regulation.

Adult↗

PubMed1997

Vinculin is associated with the E-cadherin adhesion complex.

Cadherins mediate calcium-dependent cell-cell adhesion, and this activity is regulated by cytoplasmic interactions between cadherins, catenins, and the actin-based cytoskeleton. alpha-Catenin plays a critical role in the transmembrane anchorage of cadherins, and deletion of alpha-catenin has been shown to inactivate cadherin-mediated adhesion, resulting in a nonadhesive phenotype. Here we show that serum starvation increases E-cadherin expression and induces E-cadherin-dependent adhesion in the MDA-MB-468 breast cancer cell line. This adhesion occurred despite a lack of alpha-catenin expression, which was caused by mutations in the alpha-catenin gene. Coprecipitation analysis suggests that this adhesion may be mediated by cytoplasmic connections from cadherins to the cytoskeleton involving vinculin. A high level of vinculin associated with E-cadherin immunoprecipitates was observed in MDA-MB-468 cells. In contrast, vinculin was not detected in E-cadherin complexes in the A431 and MCF-7 epithelial carcinoma cell lines, which express alpha-catenin. However, in reciprocal immunoprecipitations using anti-vinculin antibodies, E-cadherin associated strongly with vinculin in MDA-MB-468 cells and, to a lesser extent, in A431 and MCF-7 cells. These results suggest that both alpha-catenin and vinculin may be present in the adhesion complex. To test the hypothesis that vinculin associates with E-cadherin complexes via beta-catenin, excess recombinant beta-catenin or alpha-catenin fusion protein was added to MDA-MB-468 cell lysates. Both specifically inhibited the coprecipitation of E-cadherin with vinculin, suggesting competition for the same binding site. These results suggest that vinculin plays a role in the establishment or regulation of the cadherin-based cell adhesion complex by direct interaction with beta-catenin.

Amino Acid Sequence↗

PubMed1997

The cell adhesion molecule, E-cadherin, distinguishes mesothelial cells from carcinoma cells in fluids.

BACKGROUND: The distinction between benign reactive mesothelial cells and well differentiated carcinoma can be difficult in pleural, peritoneal, and especially pericardial fluids. E-cadherin is an adhesion protein that is specifically expressed in cells of epithelial lineage. In this study, anti-E-cadherin antibodies were used to identify and distinguish carcinoma cells from reactive mesothelial cells. METHODS: Pleural, peritoneal, and pericardial fluids were prepared using the Cytyc Thin Prep processor. The specimens were comprised of a mix of 45 cases that were diagnosed as carcinoma, suspicious, or reactive by Papanicolaou staining of routine material seen by the authors' service. Routine immunologic techniques were used with a commercially available E-cadherin antibody. RESULTS: In most cases of carcinoma, tumor cells showed a strong positive membranous reaction product (32 of 37). This included four cases that were not cytomorphologically diagnosed as malignant, but subsequently proved to be malignant. E-cadherin staining was not observed in five tumors, two of which were not expected to express this protein. One benign case showed cells staining for E-cadherin, although the cells were not malignant by morphologic criteria. Because this case was a surgical pelvic washing, these cells more likely were epithelial contaminants than true false-positives. CONCLUSIONS: The epithelial specific cell-cell adhesion marker E-cadherin reliably distinguishes reactive mesothelial cells from carcinoma and is a useful adjunctive test to distinguish benign reactive mesothelial cells from well differentiated carcinoma cells in fluid specimens.

Adenocarcinoma↗

PubMed1997

Alpha-catenin can form asymmetric homodimeric complexes and/or heterodimeric complexes with beta-catenin.

The cadherin-based transmembrane cell-cell adhesive complex is thought to be composed of a cadherin molecule, a beta-catenin, and an alpha-catenin, which connects the complex to the cytoskeleton. The precise stoichiometry of this complex remains uncertain. We have used a series of recombinant molecules and biophysical techniques to assess the multimeric state of human alpha- and beta-catenin in vitro and then visualized them by electron microscopy after rotary shadowing. Calculated solution molecular masses are 213 kDa for alpha-catenin, 73 kDa for beta-catenin, and 186 kDa for both. This suggests that alpha-catenin exists as a homodimer in solution, beta-catenin is a monomer, and when both are present, they form alpha/beta-catenin heterodimers. Co-precipitation and surface plasmon resonance assays localize the site of alpha-catenin dimerization to the NH2-terminal 228 amino acids. This region encompasses a high-affinity (Kd = 100 nM) binding site for beta-catenin that lies between residues 54 and 157. We anticipate that the oligomeric state of alpha-catenin and the relative stoichiometry of the components in the membrane adhesion complex will be dynamic and regulated by beta-catenin, cell adhesion, and probably other factors as well.

Binding Sites↗

PubMed1997

Comparison of the costs of fine-needle aspiration and open surgical biopsy as methods for obtaining a pathologic diagnosis.

BACKGROUND: A pathologic diagnosis is usually required to determine definitive management for a palpable lesion. In this era of cost control, fine-needle aspiration (FNA) provides a low-cost alternative to open excisional biopsy. Using a broad range of cases collected over 20 years, the authors of this study sought to quantify the savings resulting from the use of FNA on superficial palpable lesions to obtain a pathologic diagnosis. METHODS: 12,452 cases collected by the cytopathology service at the Medical College of Virginia during the period 1972-1991 were used to produce a profile of case type, diagnosis, and indication for surgery. Charge-based cost estimations or Relative Value Units were calculated using the 1995 Physicians' Fee Reference or published Medicare participant fees. The charges for the FNA procedure and open surgical biopsy were compared, and all other biopsy-related costs were omitted. RESULTS: FNA provided a sufficient pathologic diagnosis to obviate open surgical biopsy in 63-85% of the cases. Estimation of cost savings on the basis of the distribution of cases and indications for surgery suggested a savings of $250,000 to $750,000 per 1000 FNA performed, or approximately 5500 Relative Value Units. CONCLUSIONS: This study quantifies the substantial savings that result from obtaining a pathologic diagnosis by the FNA procedure rather than open surgical biopsy.

Biopsy↗

PubMed1997

Atypical squamous cells of undetermined significance: correlative histologic and follow-up studies from an academic medical center.

The diagnosis of ASCUS (atypical squamous cells of undetermined significance) was introduced in the 1988 Bethesda System for reporting cervical/vaginal cytologic findings. Outcome and appropriate management of patients with this diagnosis is not presently established. Criteria defining ASCUS are nuclear enlargement (2.5-3.0 times normal intermediate cell nucleus), mild nuclear hyperchromasia, smooth nuclear outlines with mild variation in nuclear size and shape, or else two, but not all three, cytologic criteria for human papilloma virus (HPV) cytopathic effect. All 668 cases reported as ASCUS from February 1992-December 1993 from our cytology laboratory were reviewed. These ASCUS cases represented 4.5% of all gynecologic cases diagnosed in that same time period. Of these, 284 (41%) had a subsequent colposcopic biopsy and/or endocervical curettage. The biopsied cases included 101 (36%) with condylomata, 38 (13%) with cervical intraepithelial neoplasia (CIN) I, 17 (6%) with CIN II, and 9 (3%) with CIN III. No cases of carcinoma were detected. Of patients with a cytologic diagnosis of ASCUS and subsequent cervical biopsy, 49% had low-grade cervical intraepithelial neoplasia (LGSIL), either condyloma or CIN I. Nine percent had high-grade cervical intraepithelial neoplasia, either CIN II or CIN III. These findings indicate that ASCUS defines cytologically a group of patients who may have either a concurrent or subsequent development of a squamous intraepithelial lesion (SIL). This forms a high-risk group. The management of cases with a cytologic diagnosis of ASCUS should be at least as aggressive as that of LGSIL.

Adolescent↗

PubMed1997

Degree of dysplasia following diagnosis of atypical squamous cells of undetermined significance is influenced by patient history and type of follow-up.

Previous studies have shown that atypical squamous cells of undetermined significance (ASCUS) are a predictor of higher grade lesions when patients are followed up by biopsy. The purpose of this study was to examine follow-up on all patients with ASCUS to determine if the prediction of higher grade lesions seen in association with ASCUS is a function of bias in selection of patients for biopsy. The diagnosis of ASCUS, based on Bethesda System criteria, was made on 235 cases between June 1993 and December 1994 (2% of the total cases at Yale-New Haven clinics). Of these cases, 36 were biopsied and 94 were followed by cervical/vaginal smears (CVS). As has been seen in other retrospective studies, we found that 55% (20 of 36) biopsied after ASCUS on CVS showed condyloma or dysplasia. In cases with CVS follow-up instead of biopsy, only 26% (25 of 94) had significant lesions. We find that if ASCUS is evaluated by biopsy, the prediction for higher grade lesions is roughly twice that predicted by follow-up CVS. We purpose that nonstandardized management by gynecologists results, in many instances, in selection of patients with significant past history for biopsy. Consequently, the current literature finding of ASCUS as predictor of higher grade lesions should be reanalyzed, taking into account other parameters (past history, age, etc.) in the process of defining the clinical and biological implications of the ASCUS diagnosis.

Bias↗

PubMed1997

Transcriptional defects underlie loss of E-cadherin expression in breast cancer.

Decreased expression of E-cadherin (E-cad), a calcium-dependent cell adhesion molecule, has been seen in many different epithelial cancers. Although somatic mutations in the E-cad gene have been identified in a small subset of tumors, in the majority of cancers, the mechanisms underlying loss of E-cad expression are poorly understood. We have cloned the human E-cad promoter and defined its critical components in functional assays. In eight human breast cancer cell lines, there was a striking correlation between endogenous E-cad gene expression and E-cad promoter activity observed following the introduction of reporter gene constructs into the lines. These and other observations suggest that defects in trans-acting pathways regulation E-cad expression are the primary basis for the loss of its expression in most breast cancers. The results have significant implications for understanding the gene expression differences that underlie tumor heterogeneity and progression events in breast and other epithelial cancers.

Antimetabolites, Antineoplastic↗

PubMed1997

Localization and quantitation of expression of the cell motility-related protein thymosin beta15 in human breast tissue.

Thymosin beta15 is a newly discovered 5300-Da protein that binds actin monomers and inhibits actin polymerization and might thus increase cellular motility. Thymosin beta15 is upregulated at both the mRNA and protein levels in prostate cell lines in a manner directly related to their capacity to metastasize. We hypothesize that because this protein is upregulated in cells with a propensity to metastasize, it might be a useful prognostic marker in breast cancer. Because this is a newly described protein, neither the subcellular localization of thymosin beta15 or its expression in breast cancer has been examined. We describe the use of an affinity-purified polyclonal antibody to show that within breast epithelium, thymosin beta15 is localized diffusely throughout the cytoplasm and that thymosin beta15 is upregulated in malignant (compared with benign) breast tissue. In contrast to the prostate model, thymosin beta15 is upregulated in nonmetastatic breast cancer and even ductal carcinoma in situ (compared with benign breast tissue), and, consequently, it might represent a potential early marker for breast malignancy. Additional studies are needed to evaluate the precise role and prognostic value of thymosin beta15 in breast cancer.

Adolescent↗