Disruption of the developmentally regulated Rev3l gene causes embryonic lethality.

The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.

Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145.

A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (-508 to -501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and compatible protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer.

Peroxidase-labelled lectin binding of human extravillous trophoblast.

The binding of four peroxidase-conjugated lectins, concanavalin A (Con-A), wheat germ agglutinin (WGA), peanut agglutinin (PNA) and soybean agglutinin (SBA), in both fixed and frozen tissue sections of human extravillous trophoblast (EVT) was determined. On the basis of its lectin binding properties the EVT cell population was found to be heterogeneous. PNA and SBA did not bind to any of the EVT cells. Con-A and WGA bound to most EVT cells, with the exception of the trophoblast of the chorion laeve. The trophoblast giant cells bound only Con-A and not WGA. The villous cytotrophoblast, from which the EVT cells are said to derive, does not express the sugar groups detected by the above lectin probes. The expression of mannosylated and di-N-acetylchitobiosyl residues by a fetally derived cell invites speculation that such expression enables it both to invade host maternal tissues and to avoid any adverse host immunological response.

Role of the Bcl-2 gene family in prostate cancer progression and its implications for therapeutic intervention.

Prostate cancer (PC) is an escalating health burden in the western world. A large number of patients still present with extraprostatic (i.e., T3/T4, N0, M0/M1 or any T category and M1 disease or involved lymph nodes) and therefore incurable disease. Since the work of Huggins in 1940, there have been no major therapeutic advances and androgen ablation remains the best treatment option for extraprostatic androgen-responsive PC. Eighty to ninety percent of PC patients respond well to this form of treatment initially. After a median time of approximately 2 years, however, relapse to an androgen-independent (AI) state occurs, followed by death after a further median 6 months. Androgen ablation is rarely curative. The major molecular defect in extraprostatic and AI PC is the inability of PC cells to initiate apoptosis in response to a variety of stimuli, including different forms of androgen ablation and cytotoxic agents. The balance between cellular proliferation and cell death is regulated by multiple genes or families of genes through the cell cycle. The exact mechanisms governing this intricate and complex process are as yet not fully understood. One family of genes involved in cell survival/death control is the Bcl-2 gene family, which consists of homologous proteins that function to regulate distal and crucial commitment steps of the apoptotic pathway. The Bcl-2 family constitutes both agonists and antagonists of apoptosis that function at least in part through protein-protein interactions between various members of the family. The final outcome depends on the relative ratio of death agonists and antagonists. Bcl-2 expression has been closely associated with the AI phenotype of PC. Cytotoxic chemotherapy may be used as palliative therapy in AI PC but has not been found effective. Most chemotherapeutic cytotoxic agents induce apoptosis in cancer cells by direct and indirect action on the cell cycle. In vitro and in vivo studies have established that Bcl-2 expression confers an antiapoptotic activity against androgen withdrawal and cytotoxic chemotherapy. It thus offers a tempting potential target for therapeutic manipulations of PC.

Immunolocalization of criptoregulin and amphiregulin in rectal-cancer - correlation with prognosis.

We have examined the expression of two newly identified members of the EGF family, cripto and amphiregulin (AR), in a series of 58 primary rectal carcinomas and adjacent non-involved mucosa by immunohistochemical staining using rabbit polyclonal antibodies. More than 90% (53/58) of rectal carcinomas showed AR immunoreactivity whereas cripto was found in 41 out of 58 (71%) tumours. Cripto immunoreactivity was most frequently seen in tumours arising in the lower third of the rectum (p<0.01) and in flat and excavated lesions (p<0.05). Out of 54 normal rectal mucosae adjacent to carcinoma 20 (37%) showed cripto immunoreactivity and all showed a trend towards a higher recurrence rate. Ten of these 20 (50%) rectal tumours showed less cripto immuno-reactivity than the adjacent normal mucosa and were penetrating through the bowel wall and recurred within 5 years. There was a correlation between increased cripto immunoreactivity in the normal mucosa and lymph node involvement (p=0.01). AR immunoreactivity was present in the majority (52/58, 94%) of the normal mucosae adjacent to tumours. No correlation was found between AR immunostaining, histology and prognosis.

bcl-2: role in epithelial differentiation and oncogenesis.

The precise regulation and maintenance of balance between cell proliferation and cell death in multicellular organisms is critical for tissue homeostasis. bcl-2 initiates a new gene family involved in the regulation of cell death and survival without affecting cell proliferation. Expression of Bcl-2 has been reported in a wide range of hematopoietic cells, nonneoplastic epithelia (both hormone-responsive and nonresponsive), and epithelial malignancies. Although the major group of epithelial cells expressing Bcl-2 protein are in the proliferating zones, expression is not directly related to cell proliferation. Bcl-2 is also associated with stem cells committed to differentiation and morphogenesis. The survival advantage provided by Bcl-2 prolongs the life span of epithelial cells with differentiation potential and allows proliferation, differentiation, and morphogenesis to proceed. The gene expression in hormone-responsive organs may contribute to the sustained life of those terminally differentiated epithelial cells and a decrease in Bcl-2 levels leads to cell death by apoptosis. Overexpression of bcl-2 protects epithelial cells from death, but it is neither able to immortalize normal cells, nor to cause tumorigenic transformation of immortalized epithelial cells. Heterogeneous expression of Bcl-2 in epithelial malignancies suggests that the gene is differentially regulated. Furthermore, its expression in association with precancerous lesions suggests a role in the early stage of tumorigenesis. The effects of Bcl-2 expression on sensitivity of epithelial cells to drug, radiation, and hormone therapies vary depending on the typed of tumor. Expression of Bcl-2 is associated with resistance to hormone therapy and recurrence in prostate carcinomas, whereas in lung and breast carcinomas it is associated with a better prognosis. Studies now being performed should clarify the underlying mechanisms of differential gene regulation in different tissues and show the clinical significance of the expression of bcl-2 and other members of the bcl-2 gene family.

Reactive biliary epithelium: the product of a pluripotential stem cell compartment?

Liver parenchymal cells (hepatocytes) have a low rate of turnover, but can nevertheless mount a rapid and efficient regenerative response. However, in some cases of extreme hepatotoxicity hepatocyte proliferation is restricted or even abolished, and instead biliary epithelial cells, commonly referred to as ductular oval cells, migrate into the periportal and midzonal parenchyma. Initially these cells behave as authentic biliary epithelium with expression of the biliary cytokeratin intermediate filaments, but then show hepatocytic traits such as alpha fetoprotein and albumin synthesis. Thereafter these biliary ducts rapidly vanish to be replaced by either small hepatocytes or intestinal-type cells. The proliferation and differentiation of oval cells is probably strongly influenced by paracrine signalling from liver stellate cells. Oval cells appear to be the progeny of facultative pluripotential stem cells which have the lineage potential of uncommitted gastrointestinal stem cells; these stem cells are likely to be located in the cholangioles and small interlobular bile ducts. Oval cells thus constitute an important reserve compartment for hepatocytes when hepatocyte regeneration is compromised.

Characterization of monoclonal antibodies raised to C-terminal peptides of pS2: a major trefoil peptide and motility factor expressed in adenocarcinomas and regions of mucosal injury.

Two novel monoclonal antibodies, GE1 and GE2 raised against the C-terminal 31 and 28 amino acids of the estrogen-inducible trefoil peptide pS2, are described. Both antibodies are able to detect pS2 in formalin-fixed, paraffin-embedded tissues. Conditions are presented under which pS2 can be shown in cell lines by immunohistochemistry that has previously been problematic. The antibodies can specifically show the presence of pS2 in cell lysates by Western blotting and immunoprecipitation. In the form of an affinity column, the GE1 monoclonal antibody can be used to purify pS2 from MCF-7 supernatants. The eluted peptide from the GE1 affinity column shows a single band at 6,600 Da (predicted size for pS2) on Western blotting. These antibodies are valuable reagents in the analysis of the role of trefoil peptides in the maintenance of mucosal integrity, and may have applications in the assessment of pS2 expression in chronic gastrointestinal ulceration and adenocarcinomas that secrete pS2, where it may serve as a prognostic marker.

MT1-MMP and MMP-2 mRNA expression in human ovarian tumors: possible implications for the role of desmoplastic fibroblasts.

Expression of activated MMP-2 (72 kDa type IV collagenase) is highly associated with the malignant phenotype in adenocarcinomas, but predominant expression of the mRNA appears to be in stromal cells. MT1-MMP (membrane type 1-matrix metalloproteinase) is implicated in tumor-epithelial cell surface activation of latent pro-MMP-2, indicating a mechanism for tumor-stromal interaction in invasion. We determined the relative mRNA distribution of these MMPs in human ovarian tumors with a view to analyzing potential variations in the epithelial-mesenchymal interactions dictating ovarian tumor cell spread. In situ hybridization using 35S-labeled riboprobes was used to analyze 33 human ovarian tumors and mouse xenografts of human ovarian (DOV 13, SKOV3) and breast (MCF 7) tumor cell lines known to express MT1-MMP and MMP-2. MMP-2 mRNA was expressed in 31 of 33 and MT1-MMP mRNA was expressed in 29 of 33 tumor cases. MMP-2 mRNA was predominantly expressed in desmoplastic fibroblasts and in the subepithelial stroma. MT1-MMP mRNA showed some colocalization with MMP-2 in stromal cells. Neoplastic epithelial cell labeling for MT1-MMP mRNA was present in borderline and malignant tumors but not in benign tumors, and was invariably less than stromal labeling. Xenografts of DOV 13, SKOV 3, and MCF 7 cells showed some stromal localization of MMP-2 mRNA and weak labeling of DOV 13 cells. There was variable labeling for MT1-MMP mRNA in the neoplastic cells only. The colocalization of MT1-MMP and MMP-2 mRNAs in ovarian carcinoma stroma supports the view that MT1-MMP is closely associated with MMP-2 expression and function. It suggests that either additional mechanisms are involved in regulating MMP-2 activation at the tumor cell surface, or more intriguingly, that desmoplastic fibroblasts may be the primary mediators of extracellular matrix remodeling with respect to this system.

Correlation between androgen receptor expression and FGF8 mRNA levels in patients with prostate cancer and benign prostatic hypertrophy.

AIM: To investigate the correlation between androgen receptor expression and fibroblast growth factor 8 (FGF8) mRNA levels. METHODS: 39 human prostate cancers and 14 benign prostatic hypertrophy specimens were examined immunohistochemically for androgen receptor expression and by in situ hybridisation and reverse transcription polymerase chain reaction for FGF8 expression. RESULTS: In 39 tumours there was a statistically significant negative correlation between tumour grade and FGF8 expression and a positive correlation between FGF8 and androgen receptor expression. All 14 benign hypertrophy specimens expressed moderate to high levels of FGF8 and androgen receptor. CONCLUSIONS: Loss of FGF8 may be a factor involved in the development of prostatic cancer.

Immunohistochemical analysis of p53 oncoprotein and proliferating cell nuclear antigen (PCNA) in the cervix uteri.

Recent data suggest an inverse correlation between human papillomavirus (HPV) infection and p53 tumor-suppressor gene mutation. In an attempt to elucidate the role of p53 mutations in cervical neoplasias, 65 cervical lesions, ranging from normal to malignant, were examined for overexpression of p53 protein by immunohistochemistry in paraffin-embedded tissue, and correlated with proliferating cell nuclear antigen (PCNA). An overexpression was seen in 35% of well-differentiated and 32.5% of poorly-differentiated squamous carcinomas, in 43.33% of microinvasive and 21.66% of CIS. More than 50% of neoplastic cells were immunoreactive for p53 protein in 10% of well-differentiated squamous carcinomas. Other positive specimens showed reactivity in < 24% of tumor cells. No staining was found in adenocarcinoma, dysplastic tissue, condylomas and normal tissue (83.07%). In contrast PCNA was detected in all cases of invasive squamous carcinomas, adenocarcinoma, CIS, CIN III, in 32.5% of CIN II, in 29.54% CIN I, and in 53.52% of wart. More than 50% of tumor cells showed nuclear staining for PCNA protein in 61.17% of invasive squamous carcinomas, in 21.66% of CIS, in 39% CIN III, in 32.5% CIN II and in 7.64% of wart. In the remain cases the positivity of nuclear staining was < 24%. No staining was present in 20% of cases including in normal cervix. Our data suggest that the viral-host protein interactions result in loss of the negative growth control normally exerted by p53. The consequence of HPV infection is a loss of functional wild-type p53 protein within the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Trefoil peptide expression in intestinal adaptation and renewal.

There are many avenues where molecular biology is important in studying the gut, and here we explore methods for defining expression of a new gene family in the gut. We have defined the pattern of trefoil peptide gene expression in the ulceration-associated cell lineage (UACL) and in the nearby mucosa in Crohn's disease. In the UACL, human spasmolytic polypeptide mRNA and peptide are expressed in the acinar and proximal duct cells, whereas pS2 mRNA and peptide are found in the distal duct cells and in the surface cells. In adjacent mucosa, pS2 mRNA and protein are expressed ectopically by goblet cells. Ultrastructural immunolocalisation showed the pS2 to be co-packaged in the mucous cell granules. pS2 peptide was demonstrated in local neuroendocrine cells and was also co-packaged with the neuroendocrine granules. The crypts associated with the UACL also showed marked neuroendocrine cell hyperplasia. We have also cloned the newest trefoil peptide intestinal trefoil factor from human and rat intestinal mucosa and shown its co-expression with mucus by normal intestinal goblet cells. The co-packaging of the same secretory protein in both mucous and neuroendocrine granules, which have different functions, is unusual and indicates an important role for pS2 in the secretory process itself or as a ligand delivered to its receptor via multiple routes. We conclude that the trefoil peptides are widely distributed in the intestine in inflammatory bowel disease and are of considerable potential functional importance.

The effects of sex steroid hormones and interleukin-1-beta on MUC1 expression in endometrial epithelial cell lines.

Oestrogen, progesterone and paracrine signals from the embryo have been associated with the overall control of implantation. Changes in the expression of the heavily glycosylated transmembrane glycoprotein MUC1 mucin on the endometrial epithelium are also thought to be important for embryo attachment. Increased MUC1 expression has been correlated with elevated progesterone levels in the secretory phase of the menstrual cycle. Embryonic control of endometrial receptivity through changes in MUC1 expression could be achieved through the interleukin-1 system. Four endometrial epithelial cell lines (HEC1A, HEC1B, Ishikawa and RL592) were treated with oestrogen and progesterone (with or without interleukin-1-beta) and were subjected to immunocytochemistry and flow cytometric analysis to determine MUC1 production using MUC1 antibodies. HEC1A (oestrogen receptor (ER) and progesterone receptor (PR) positive) and HEC1B (ER positive and PR negative) were transfected with the MUC1 promoter, underwent similar treatment regimes and the activity of the MUC1 promoter relative to their untreated controls was determined using a chloramphenicol acetyltransferase (CAT) enzyme-linked immunoassay. Using the cell lines, we determined that endometrial MUC1 expression is up-regulated by progesterone, consistent with the in vivo increases in MUC1 related to high progesterone levels. We also revealed that neither oestrogen, nor interleukin-1-beta, appear to modulate MUC1. Progesterone-dependent regulation of MUC1 is likely to be an important factor in determining endometrial receptivity.

Brief report: no evidence for parvovirus B19 or hepatitis E virus as a cause of acute liver failure.

Viral hepatitis A and B are known to cause acute liver failure. While nearly 20% of acute liver failure cases are of indeterminate etiology, screening for other viruses has not been uniformly performed. We looked for evidence for parvovirus B19 and hepatitis E virus in sera from U.S. acute liver failure patients. For B19, 78 patients' sera, including 34 with indeterminate etiology, were evaluated by DNA dot-blot hybridization, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay for immunoglobin G and M antibodies; none showed evidence for infection.

The expression pattern of MUC1 glycoforms and other biomarkers of endometrial receptivity in fertile and infertile women.

Changes in the surface epithelium of the endometrium, characterized in part by alterations in cell-surface molecules, sex steroid receptors and the appearance of pinopodes, coincide with the window of endometrial receptivity in the menstrual cycle. This study was performed to evaluate the usefulness of hematoxylin and eosin staining, scanning and transmission microscopy, and MUC1 glycoform, sex steroid receptor, and interleukin receptor (type 1) expression as biomarkers of endometrial receptivity using carefully characterized clinical fertile and infertile groups of women. Using a combination of immunohistochemistry and scanning electron microscopy (SEM) called scanning immunoelectron microscopy (SIM), we confirmed that MUC1 mucin was not associated with the endometrial pinopodes, which have been linked with embryo adhesion. We also showed that failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin, and retention of nuclear progesterone receptor (PR) particularly in epithelial cells. Hematoxylin and eosin staining, transmission electron microscopy (TEM), SEM in isolation and immunohistochemistry for interleukin receptor were not shown to be useful markers. Progesterone-dependent regulation of MUC1 appears to be an important factor in determining endometrial receptivity.

Stimulation of defective Gunn-rat liver uridine diphosphate glucuronyltransferase activity in vitro by alkyl ketones.

Addition of alkyl ketone (10mM) to Gunn-rat liver homogenates increased UDP-glucuronyltransferase activity towards 2-aminophenol by 10--20 fold, up to enhanced values of enzyme activity observed with similarly treated Wistar-rat liver homogenates. Alkyl ketones also activate the defective enzyme purified from Gunn-rat liver. This genetic deficiency of UDP-glucuronyltransferase activity is no longer apparent when assayed in the presence of alkyl ketones.

Microwave-stimulated immunogold silver staining.

The effect of microwave-generated energy for the demonstration of immunoglobulins in periodate lysine paraformaldehyde dichromate fixed paraffin sections of human reactive tonsil using the immunogold silver staining technique has been evaluated. Results of the study show that microwave stimulation permits incubation times of both the primary antibody and the immunogold reagent to be greatly reduced. Specific staining of equal intensity to that produced using the longer standard procedure together with minimal background staining is achieved.

Prostate cancer; the interface between pathology and basic scientific research.

Current research into prostate cancer increasingly demands greater input from pathologists. There is a requirement for improved morphological assessment, classification and grading of neoplasia. The provision of optimally preserved material and establishment of tissue 'banks' is vital to facilitate molecular biological analysis. Microdissection of archival material can provide a source of relatively pure DNA and mRNA which can 'be further amplified by PCR/RT-PCR. This enables allelic imbalance, point mutations and other genetic abnormalities to be demonstrated. In-situ hybridization for mRNA is feasible on fixed tissues and allows precise localization of gene expression on complex tissues or for labile gene products. Experimental models including transgenic mice and in-vitro co-culture systems require sophisticated morphological analysis. Experts in morphological analysis are essential members of basic scientific and translational research teams.