ShriHari S. Kadkol

Genetic, immunohistochemical, and clinical features of medullary carcinoma of the pancreas: A newly described and characterized entity.

Medullary carcinomas of the pancreas are a recently described, histologically distinct subset of poorly differentiated adenocarcinomas that may have a unique pathogenesis and clinical course. To further evaluate these neoplasms, we studied genetic, pathological, and clinical features of 13 newly identified medullary carcinomas of the pancreas. Nine (69%) of these had wild-type K-ras genes, and one had microsatellite instability (MSI). This MSI medullary carcinoma, along with three previously reported MSI medullary carcinomas, were examined immunohistochemically for Mlh1 and Msh2 expression, and all four expressed Msh2 but did not express Mlh1. In contrast, all of the medullary carcinomas without MSI expressed both Msh2 and Mlh1. Remarkably, the MSI medullary carcinoma of the pancreas in the present series arose in a patient with a synchronous but histologically distinct cecal carcinoma that also had MSI and did not express Mlh1. The synchronous occurrence of two MSI carcinomas suggests an inherited basis for the development of these carcinomas. Indeed, the medullary phenotype, irrespective of MSI, was highly associated with a family history of cancer in first-degree relatives (P < 0.001). Finally, one medullary carcinoma with lymphoepithelioma-like features contained Epstein-Barr virus-encoded RNA-1 by in situ hybridization. Therefore, because of medullary carcinomas special genetic, immunohistochemical, and clinical features, recognition of the medullary variant of pancreatic adenocarcinoma is important. Only by classifying medullary carcinoma as special subset of adenocarcinoma can we hope to further elucidate its unique pathogenesis.

Tumor suppression and potentiation by manipulation of pp32 expression.

Alternative use of genes of the closely-related pp32 family is a common occurrence in human prostate cancer. pp32r1 and pp32r2, the oncogenic members of the pp32 family, are expressed in prostatic adenocarcinoma, while adjacent benign prostate continues to express pp32. This study focuses upon the role of pp32 in tumor suppression. We demonstrate that antisense inhibition of pp32 in NIH3T3 cells leads to a variety of phenotypic changes associated with transformation including reduced serum dependence and loss of contact inhibition. NIH3T3 cells with antisense-inhibited pp32 are not tumorigenic, but are markedly more susceptible to oncogenic stimuli such as ras. In contrast, constitutive expression of pp32 abolishes ras mediated transformation in vitro and tumorigenesis in vivo. These data demonstrate, from the functional aspect, that pp32 acts as a tumor suppressor. Furthermore, inactivation of pp32 function through alternative gene use may be a critical event in tumor evolution and progression.

C6 Produced by Macrophages Contributes to Cardiac Allograft Rejection

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.AaBu) cardiac allografts by PVG.1U (RT1.AuBu) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG.R8 (C6−) donors to PVG.1U (C6−) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6−) recipients of PVG.R8 (C6−) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6−) recipients that were reconstituted with bone marrow from PVG.1U (C6−) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.

Novel nuclear phosphoprotein pp32 is highly expressed in intermediate- and high-grade prostate cancer

pp32 is a differentiation‐regulated nuclear phosphoprotein that is highly expressed in many cancers, but is restricted to self‐renewing and long‐lived normal cell populations. During murine embryogenesis, pp32 is expressed in primitive cell populations, diminishing as tissues terminally differentiate. Functionally, pp32 confers resistance to programmed cell death and, paradoxically, inhibits transformation mediated in vitro by a broad range of oncogenes, suggesting that pp32 is a multifunctional molecule with potentially complex activities in cancer.

Identification of sequences required for inhibition of oncogene-mediated transformation by pp32

Oncogenic potential in prostate cancer is modulated in part by alternative use of genes of the pp32family. This family includes the tumor suppressor pp32, expressed in normal tissue, and the pro-oncogenic genespp32r1 and pp32r2 that are found principally in neoplastic cells. At the protein level, pp32, pp32r1, and pp32r2 are approximately 90% identical, yet they subsume opposite functions. In this study, we identify the region of pp32 associated with the ability to inhibit oncogene-mediated transformation in a rat embryo fibroblast system, an in vitro correlate of tumor-suppressive activity. Deletion and truncation analysis define a region spanning pp32 amino acids 150–174 as absolutely required for inhibition of transformed foci elicited by RAS and MYC. Comparison of pp32 with the pp32r1 sequence by moving averages of sequence identity reveals divergence over this region; pp32r2 also differs in this region through truncation after pp32 amino acid 131. The deletion experiments and the experiments of nature therefore converge to demonstrate that tumor-suppressive functions of pp32 reside in amino acids 150–174. Identification of this minimal tumor-suppressive region should help elaborate the pathways and mechanisms through which pp32 family members exert their functions.

Feasibility of implementing a comprehensive warfarin pharmacogenetics service.

To determine the procedural feasibility of a pharmacist‐led interdisciplinary service for providing genotype‐guided warfarin dosing for hospitalized patients newly starting warfarin.

In situ hybridization—Theory and practice

In situ hybridization is a technique to determine and localize target nucleic acids in morphologically preserved tissue sections. Recent advances in methods have greatly increased the sensitivity of the technique, and it is currently possible to detect extremely few copies of any given target sequence with nonisotopic methods. In this teaching review, we integrate theoretical background, technical considerations, and guidelines for usage for this important component of molecular diagnosis.

Authenticating Cell Lines in Ophthalmic Research Laboratories

Authentication of cell lines in biomedical research has been elevated to a very high priority. From a review of the literature, Lacroix1 reviewed the issue of cross-contamination of cell lines including the well known contamination of cell lines with HeLa cells,2 and the mis-identification of the ECV304 cell line as “immortalized endothelial cells” when these cells in fact originated from T24 bladder carcinoma cells.3 Lacroix 1 estimated that between 18 and 36% of cell lines have been misclassified. One survey at a large research institution suggested that fewer than 50% of researchers authenticate their cell lines.4 Nardone5 proposed recently that identification of cell lines be required of investigators before grants are awarded, and the National Institutes of Health subsequently called for researchers to authenticate cell lines as a prerequisite for grant funding.6

AML1-ETO Decreases ETO-2 (MTG16) Interactions with Nuclear Receptor Corepressor, an Effect That Impairs Granulocyte Differentiation

The t(8;21) chromosome abnormality in acute myeloid leukemia targets the AML1 and ETO genes to produce the leukemia fusion protein AML1-ETO. Another member of the ETO family, ETO-2/MTG16, is highly expressed in murine and human hematopoietic cells, bears >75% homology to ETO, and like ETO, contains a conserved MYND domain that interacts with the nuclear receptor corepressor (N-CoR). AML1-ETO prevents granulocyte but not macrophage differentiation of murine 32Dcl3 granulocyte/macrophage progenitors. One possible mechanism is recruitment of N-CoR to aberrantly repress AML1 target genes. We wished to examine another mechanism by which AML1-ETO might impair granulocyte differentiation. We demonstrate that AML1-ETO decreases interactions between ETO-2 and N-CoR. Furthermore, overexpression of ETO-2 relieves AML1-ETO-induced granulocyte differentiation arrest. This suggests that decreased interactions between ETO-2 and N-CoR may contribute to granulocyte differentiation impairment. The MYND domain coimmunoprecipitates with N-CoR and inhibits interactions between ETO-2 and N-CoR, presumably by occupying the ETO-2 binding site on N-CoR. This inhibition of ETO-2 interactions with N-CoR is specific because the MYND domain does not inhibit retinoic acid receptor interactions with N-CoR. To examine the effect of decreasing interactions between ETO-2 and N-CoR in hematopoietic cells, without effects of AML1-ETO such as direct repression of AML1 target genes, the MYND domain was expressed in 32Dcl3 and human CD34+ cells. The MYND domain prevented granulocyte but not macrophage differentiation of both 32Dcl3 and human CD34+ cells, recapitulating this effect of AML1-ETO. In conclusion, decreasing interactions between ETO-2 and N-CoR, an effect of AML1-ETO, inhibits granulocyte differentiation.

Increased neurotensin receptor-1 expression during progression of colonic adenocarcinoma

The high affinity neurotensin receptor (NTSR1) mediates most of the biologic effects of neurotensin (NT), a 13-amino acid peptide that stimulates growth in certain cell types. NT is expressed in fetal but not differentiated colonic epithelium and is re-expressed in colonic adenocarcinoma. The cognate receptor, NTSR1, is also not expressed or is present at a low level in adult colonic epithelial cells but is expressed in most colon cancer cell lines. These observations suggest that altered NT-NTSR1 signaling may be associated with malignant transformation in the colon. To further understand the possible role of NTSR1 expression in colonic tumorigenesis and progression, we examined NTSR1 mRNA by in situ hybridization in normal colonic mucosa, adenomas, and colonic adenocarcinomas. NTSR1 mRNA expression was undetectable or weak in superficial differentiated epithelial cells of normal colonic epithelium, but adenomas and adenocarcinomas showed moderate to strong expression (p<0.05). Adenocarcinomas showed a higher level of expression compared to adenomas (p<0.05). Furthermore, adenocarcinomas that infiltrated into and beyond the muscularis propria showed a higher intensity of NTSR1 expression compared with tumors that were localized to the mucosa or submucosa. In some cases, infiltrating margins and foci of lymphovascular invasion showed a higher intensity of expression than the main mass of the tumor. These results suggest that increased NTSR1 expression may be an early event during colonic tumorigenesis and also contribute to tumor progression and aggressive behavior in colonic adenocarcinomas. NTSR1 may thus be a potential target for preventive or therapeutic strategies in colon cancer.