Reverse Transciptase-polymerase Chain Reaction Research Articles

Antitumor Efficacy Of DMSA Modified Fe3O4magnetic Nanoparticles Combined With Arsenic Trioxide and Adriamycin In Jurket Cells

The objective of the present study was to investigate the anticancer efficacy of dimercaptosuccinic acid modified iron oxide (DMSA-Fe3O4) magnetic nanoparticles (MNPs) combined with arsenic trioxide (As2O3) and doxorubicin (ADM) in non-Hodgkin's lymphoma (NHL) cell line (Jurket cells). The growth inhibition rate of Jurket cells was determined by MTT assay. Characteristics of DMSA-Fe3O4MNPs and distribution of nanoparticles taken up by Jurket cells were observed under a transmission electron microscopy (TEM). Further, apoptosis of cells and intracellular concentration of ADM were detected by flow cytometry (FCM). DAPI staining was used to view apoptotic cellular morphology. Subsequently, transcription and protein expression levels of bcl-2, NF¦ÊB, survivin, bax, p53 and caspase-3 were determined by reverse transciptase polymerase chain reaction (RT-PCR) and Western blotting analysis, respectively. The results of MTT assay indicated that the inhibition of Jurket cells by the combined form of ADM and As2O3 was significantly higher than either ADM or As2O3 alone. However, ADM-As2O3 MNPs proved superior over all other groups. TEM observation revealed that the majority of MNPs were quasi-spherical with an average diameter of about 18 nm and the MNPs taken up by cells were located in the endosome vesicles of cytoplasm. The apoptotic rate and accumulation of intracellular ADM in ADM-As2O3 MNPs group were significantly higher than those in control, ADM, As2O3 and ADM + As2O3 groups. In addition, DAPI staining of Jurket cells from ADM-As2O3 MNPs group clearly exhibited more morphological changes (severe structural alterations) than other groups. Moreover, transcription and protein expression of bcl-2, NF¦ÊB, survivin, bax, p53 and caspase-3 of Jurket cells were regulated at the most remarkable extent in ADM-As2O3 MNPs group as compared with other groups. These findings suggest that the antitumor efficacy of the combination of novel ADM-As2O3 MNPs on Jurket cells would be a promising strategy for lymphoma therapy. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Promotion of monacolin K production in Monascus extractive fermentation: the variation in fungal morphology and in the expression levels of biosynthetic gene clusters.

Monacolin K, an important secondary metabolite of Monascus, possesses a cholesterol-lowering effect and is widely used in the manufacture of antihypertensive drugs. In the present study, we constructed an extractive fermentation system by adding non-ionic surfactant and acquired a high monacolin K yield. The mechanism was determined by examining both cell morphology and the transcription levels of the related mokA-I genes in the monacolin K biosynthetic gene cluster. The monacolin K yield was effectively increased to 539.59 mg L-1 during extraction, which was an increase of 386.16% compared to that in the control group fermentation. The non-ionic surfactant showed good biocompatibility with Monascus. Electron scanning microscopy revealed alterations in the morphology of Monascus. The loosened mycelial structure and increased number of cell surface wrinkles were found to be related to the increased cell-membrane permeability and extracellular accumulation of monacolin K. Gene expression levels were measured via a quantitative reverse transciptase-polymerase chain reaction. By contrast, in the control group, mokA, mokB, mokC, mokD and mokF showed higher-level and longer-term expression in the extractive fermentation group, whereas mokE and mokG did not present a similar trend. The expression levels of mokH and mokI, encoding a transcription factor and efflux pump, respectively, were also higher than the control levels. The addition of a non-ionic surfactant to Monascus fermentation effectively increases the yield of monacolin K by transforming the fungus morphology and promoting the expression of monacolin K biosynthesis genes. © 2021 Society of Chemical Industry.

Genome-wide characterization of differential transcript usage in Arabidopsis thaliana.

Alternative splicing and the usage of alternate transcription start- or stop sites allows a single gene to produce multiple transcript isoforms. Most plant genes express certain isoforms at a significantly higher level than others, but under specific conditions this expression dominance can change, resulting in a different set of dominant isoforms. These events of differential transcript usage (DTU) have been observed for thousands of Arabidopsis thaliana, Zea mays and Vitis vinifera genes, and have been linked to development and stress response. However, neither the characteristics of these genes, nor the implications of DTU on their protein coding sequences or functions, are currently well understood. Here we present a dataset of isoform dominance and DTU for all genes in the AtRTD2 reference transcriptome based on a protocol that was benchmarked on simulated data and validated through comparison with a published reverse transciptase-polymerase chain reaction panel. We report DTU events for 8148 genes across 206 public RNA-Seq samples, and find that protein sequences are affected in 22% of the cases. The observed DTU events show high consistency across replicates, and reveal reproducible patterns in response to treatment and development. We also demonstrate that genes with different evolutionary ages, expression breadths and functions show large differences in the frequency at which they undergo DTU, and in the effect that these events have on their protein sequences. Finally, we showcase how the generated dataset can be used to explore DTU events for genes of interest or to find genes with specific DTU in samples of interest.

Prostate cancer diagnosis by detection of uPM3 in urine

Background: The widespread acceptance of serum PSA testing for the early detection of prostate cancer has been hampered by the low specificity and unnecessary biopsies. Methods: This study was carried out to detect prostate cancers in patients with equivocal PSA values using RT-PCR (Reverse transciptase- polymerase chain reaction) to detect the uPM3 (urinary protein M3) in post prostatic massage urine samples. uPM3 is a mRNA released in prostate cancer patients due to the presence of the oncogene DD3 also called as PCA3 on chromosome 9. Results: There were a total of 27 cases of prostate carcinoma and 33 cases of PIN (prostate intraepithelial neoplasia) detected in this study. The sample collection accuracy depending on presence of beta2 microglobulin (housekeping gene) band was 88%. The sensitivity and specificity of uPM3 keeping histopathology as gold standard was 85.18% and 98.78% respectively. Positive predictive value was 88.46% and negative predictive value was 98.38% which is much higher than PSA. However, no significant correlation of uPM3 positivity was found with Gleason’s grade, metastatic potential and pathological stage. Conclusion: Unlike serum PSA, uPM3 was found to be independent of prostate volume thereby having no false positives unlike raised PSA because of large benign prostate hypertrophy.

Repeated examination of natural sapovirus infections in pig litters raised under experimental conditions.

BackgroundPorcine sapovirus, belonging to the family Caliciviridae, is an enteric virus that is widespread in the swine industry worldwide. A total of 14 sapovirus genogroups have been suggested and the most commonly found genogroup in swine is genogroup III (GIII). The goal of the present experiment was to examine the presence of sapovirus in 51 naturally infected pigs at two different time points. The pigs were kept under experimental conditions after weaning. Previous studies on sapovirus have primarily been of a cross sectional nature, typically prevalence studies performed on farms and abattoirs. In the present study, faecal samples, collected from each pig at 5½ weeks and 15–18 weeks of age, were analysed for sapovirus by reverse transciptase polymerase chain reaction and positive findings were genotyped by sequencing.ResultsAt 5½ weeks of age, sapovirus was detected in the majority of the pigs. Sequencing revealed four different strains in the 5½ week olds—belonging to genogroups GIII and GVII. Ten to 13 weeks later, the virus was no longer detectable from stools of infected pigs. However, at this time point 13 pigs were infected with another GIII sapovirus strain not previously detected in the pigs studied. This GIII strain was only found in pigs that, in the initial samples, were virus-negative or positive for GVII.ConclusionsAt 5 weeks of age 74 % of the pigs were infected with sapovirus. At 15–18 weeks of age all pigs had cleared their initial infection, but a new sapovirus GIII strain was detected in 25 % of the pigs. None of the pigs initially infected with the first GIII strain were reinfected with this new GIII strain, which may indicate the presence of a genogroup-specific immunity.

Human Bocavirus among Viral Causes of Infantile Gastroenteritis

Background: Human bocavirus (HBoV) infection possibly plays a role in gastroenteritis because of the frequent manifestation of gastrointestinal symptoms. Objectives: Detect human bocavirus (HBoV) and assess its prevelance among gastroenteritis associated viral agents in infants with gastroenteritis in Benha University Hospital. Methodology: The study was carried out on 100 stool samples collected from 100 infants with acute gastroenteritis for detection of Rotavirus (RV), Norovirus (NoV) & Astrovirus (AstV) by multiplex reverse transciptase polymerase chain reaction and detection of Adenovirus(AdV) & HBoV by multiplex polymerase chain reaction. Results: Viral agents were detected in 57 (57%) samples; 51 (51%) samples show mono-infection while 6 (6%) samples show co-infection. Rotavirus, Norovirus, Adenovirus and astrovirus were detected in 37%, 14%, 7.0%, and 3% of the study population, respectively; HBoV was detected in 2%. Conclusion: This percentage of HBoV suggests that it might play a minor role in gastroenteritis

Bone-Conditioned Medium Changes Gene Expression in Bone-Derived Fibroblasts.

Autologous bone is used for augmentation in the course of oral implant placement. Bone grafts release paracrine signals that can modulate mesenchymal cell differentiation in vitro. The detailed genetic response of the bone-derived fibroblasts to these paracrine signals has remained elusive. Paracrine signals accumulate in bone-conditioned medium (BCM) prepared from porcine cortical bone chips. In this study, bone-derived fibroblasts were exposed to BCM followed by a whole genome expression profiling and downstream quantitative reverse transciptase polymerase chain reaction of the most strongly regulated genes. The data show that ADM, IL11, IL33, NOX4, PRG4, and PTX3 were differentially expressed in response to BCM in bone-derived fibroblasts. The transforming growth factor beta (TGF-β) receptor 1 antagonist SB431542 blocked the effect of BCM on the expression of the gene panel, except for IL33. These in vitro results extend existing evidence that cortical bone chips release paracrine signals that provoke a robust genetic response in mesenchymal cells that is not exclusively mediated via the TGF-β receptor. The present data provide further insights into the process of graft consolidation.

Cytotoxic effect of γ-sitosterol from Kejibeling (<em>Strobilanthes crispus</em>) and its mechanism of action towards <em>c-myc</em> gene expression and apoptotic pathway

Background: This study aimed to analyze the cytotoxicity effect of γ-sitosterol isolated from “Kejibeling” (Strobilanthes crispus), a medicinal plant, on several cancer cell lines. The mechanisms of the effects were studied through the expression of cancer-caused gene, c-myc and apoptotic pathways.Methods: This in vitro study was done using human colon cancer cell lines (Caco-2), liver cancer cell lines (HepG2), hormone-dependent breast cancer cell lines (MCF-7) and the normal liver cell lines (Chang Liver). The cytotoxic effect was measured through MTT assay and the potential cytotoxic value was calculated by determining the toxic concentration which may kill up to 50% of the total cell used (IC50). Meanwhile, the cytotoxic mechanism was studied by determining the effect of adding γ-sitosterol to the c-myc gene expression by reverse transciptase-polymerase chain reaction (RT-PCR). The effect of γ-sitosterol through apoptotic pathway was studied by using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.Results: γ-sitosterol was cytotoxic against Caco-2, HepG2, and MCF-7 with IC50-values of 8.3, 21.8, and 28.8 μg/mL, respectively. There were no IC50-values obtained from this compound against Chang Liver cell line. This compound induced apotosis on Caco-2 and HepG2 cell lines and suppressed the c-myc genes expression in both cells.Conclusion: γ-sitosterol was cytotoxic against colon and liver cancer cell lines and the effect was mediated by down-regulation of c-myc expression and induction of the apoptotic pathways.

Antitumor efficacy of DMSA modified Fe3O4 magnetic nanoparticles combined with arsenic trioxide and adriamycin in Raji cells.

The objective of the present study was to investigate the anticancer efficacy of dimercaptosuccinic acid modified iron oxide (DMSA-Fe3O4) magnetic nanoparticles (MNPs) combined with arsenic trioxide (As2O3) and doxorubicin (ADM) in non-Hodgkin's lymphoma (NHL) cell line (Raji cells). The growth inhibition rate of Raji cells was determined by MTT assay. Characteristics of DMSA-Fe3O4 MNPs and distribution of nanoparticles taken up by Raji cells were observed under a transmission electron microscopy (TEM). Further, apoptosis of cells and intracellular concentration of ADM were detected by flow cytometry (FCM). DAPI staining was used to view apoptotic cellular morphology. Subsequently, transcription and protein expression levels of bcl-2, NFKB, survivin, bax, p53 and caspase-3 were determined by reverse transciptase polymerase chain reaction (RT-PCR) and Western blotting analysis, respectively. The results of MTT assay indicated that the inhibition of Raji cells by the combined form of ADM and As2O3 was significantly higher than either ADM or As2O3 alone. However, ADM-As2O3 MNPs proved superior over all other groups. TEM observation revealed that the majority of MNPs were quasi-spherical with an average diameter of about 18 nm and the MNPs taken up by cells were located in the endosome vesicles of cytoplasm. The apoptotic rate and accumulation of intracellular ADM in ADM-As2O3 MNPs group were significantly higher than those in control, ADM, As2O3 and ADM+As2O3, groups. In addition, DAPI staining of Raji cells from ADM-As,O3 MNPs group clearly exhibited more morphological changes (severe structural alterations) than other groups. Moreover, transcription and protein expression of bcl-2, NFKB, survivin, bax, p53 and caspase-3 of Raji cells were regulated at the most remarkable extent in ADM-As2O3, MNPs group as compared with other groups. These findings suggest that the antitumor efficacy of the combination of novel ADM-As2O3, MNPs on Raji cells would be a promising strategy for lymphoma therapy.

Abstract 167: MicroRNA Profiles of Non--ST-Elevation Myocardial Infarction

Significance: MicroRNAs (miRNAs) are short, stable, noncoding translational regulators used as early biomarkers for numerous disease processes. Although specific miRNA patterns are associated with STEMI, the profile and timecourse of miRNA release in NSTEMI is currently unknown but of potential clinical significance.Materials and Methods: With IRB approval, patients identified with chest pain were identified at the UMass Medical Center. Upon ED arrival (T0), a K2EDTA tube of whole blood was obtained with simultaneous troponin cTnI levels. After a 6 hour (T6) observation period, another additional K2EDTA tube was drawn, again contemporaneous with cTnI levels. Patients were selected based on an elevated cTnI level (greater then or equal to ≥ 0.04 ng/ml) at T6 as compared to a negative cTnI levels (< 0.04 ng/ml) at T0. MiRNA was isolated from patient plasma using a Trizol extraction procedure. Real-time reverse transciptase polymerase chain reaction (QRT-PCR) was performed for 94 a priori miRNAs common to cardiovascular development or disease. Results: Of the 94 miRNAs examined, 11 demonstrated greater than 2-fold change as compared to baseline (Figure 1). A subset of this cohort, namely miRNAs 208a, 483-5p, and 601 revealed statistically significant fold difference (p< 0.05). MiRNA 208a is associated with acute MI. MiRNA 601 has not been previously shown correlated with heart disease. Conclusion: This investigation reveals a novel 11 member miRNA profile, including miRNA 208a, 483-5p, and 601, which is associated with NSTEMI. Our findings suggest this miRNA profile by be used as a potential biomarker of myocardial injury.

Genomic chondrocyte culture profiling by array-CGH, interphase-FISH and RT-PCR

In vitro expansion is an important step to acquire sufficient cells in human tissue engineering technologies. The high number of chondrocytes needed for human articular cartilage implants requires in vitro expansion of the primary cells, bearing a theoretical risk of in vitro induced changes in the genomes. To gain more insights into this situation, model cultures were prepared and analyzed. 25 chondrocyte cell DNA samples from nine donors were analyzed by array comparative genomic hybridization (aCGH) on whole genome level and 28 chondrocyte cell samples from 16 individuals were analyzed by fluorescence in situ hybridization (FISH) on single cell level. The expanded cells were further characterized upon the chondrocytic mRNA phenotype by reverse-transciptase polymerase chain reaction (RT-PCR). The molecular karyotyping results revealed autosomal stability, but all male samples analyzed by aCGH displayed a variable loss of the Y-chromosome. These data were confirmed by FISH-experiments and suggest an age dependant effect toward the loss of the Y-chromosome in cultured chondrocytes. RT-PCR data for the mRNAs from collagen types I, II, and aggrecan and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) did not reveal any correlation of transcriptional activity in cultures with Y-chromosome losses, nor were there statistically significant differences between cells from female and male donors. While cells of male origin may suffer from an age-related loss of the Y-chromosome, there was no indication of a functional impairment. The data suggest some caution toward applying proliferative steps when considering chondrocytes from elderly male patients for tissue engineering approaches.

Overexpression of A disintegrin and metalloproteinase 28 is correlated with high histologic grade in conventional chondrosarcoma

Low-grade chondrosarcoma and enchondroma are occasionally difficult to differentiate solely by reference to clinicoradiologic and histologic findings. The A disintegrin and metalloproteinases are a new gene family of proteins having a metalloprotease domain with matrix metalloproteinases and play an important role in the chondrocyte development process. Therefore, we analyzed the expression of A disintegrin and metalloproteinases and matrix metalloproteinases in several kinds of cartilaginous bone tumors at the messenger RNA level and immunohistochemical protein level and ascertained their relationships to the histologic degree of malignancy. Reverse transciptase-polymerase chain reaction and real-time quantitative reverse transciptase-polymerase chain reaction of the expression of the A disintegrin and metalloproteinase family in cartilaginous bone tumors demonstrated that A disintegrin and metalloproteinase 28 messenger RNA levels in grade I chondrosarcoma were significantly higher than those in enchondroma (P = .009). Moreover, positive immunoreactivity of A disintegrin and metalloproteinase 28 was observed in 12 (59%) of 22 patients with grade I chondrosarcoma and in 21 (91%) of 23 patients with grade II chondrosarcoma, respectively. In contrast, only 2 (9%) of 21 cases of enchondromas demonstrated positive staining for A disintegrin and metalloproteinase 28. On the other hand, the immunohistochemical expressions of matrix metalloproteinase 9 and matrix metalloproteinase 13 were significantly higher in enchondromas than in normal cartilage tissue; however, there is no statistically different expression between enchondromas and grade I chondrosarcomas. We detected that overexpression of A disintegrin and metalloproteinase 28 was increased according to its histologic grade in conventional chondrosarcoma and could be one of the helpful tools in distinguishing between low-grade chondrosarcoma and enchondroma.

Attempts at immortalization of crustacean primary cell cultures using human cancer genes

Primary cell cultures from crustacea have been initiated since the 1960s, yet no permanent cell line is available. Primary cells have a limited proliferative capacity in culture due to cellular senescence, which is regulated by a group of dominant senescence genes. The aim of this research was to manipulate cell cycle regulation by transfecting Cherax quadricarinatus primary cells with oncogenes, in an effort to induce a permanent cell line. Human papillomaviruses (HPV) play a critical role in the formation of anogenital cancer. Research has demonstrated that the HPV-expressed E6 and E7 proteins function concomitantly to disrupt the p53 and retinoblastoma (Rb) tumor suppressor genes, regulators of the cell-cycle checkpoints at the first gap (G(1)) phase. HPV E6 and E7 genes were transfected into the C. quadricarinatus cells by lipofection. Successful transfection was demonstrated by the presence of oncogene messenger RNA by reverse transciptase polymerase chain reaction. At day 150, transfected cells still remain viable, although cell proliferation was stagnant. It may be that while transfection of the oncogenes was successful, no proliferation of the C. quadricarinatus cells was evident due to a lack of telomere maintenance.

Comparative study for detecting micrometastases of lymph nodes in patients with lung cancer

To set up a new method to detect occult micrometastases of lymph nodes in patients with no-small-cell lung cancer (NSCLC). We had detected 195 lymph node samples taken from 25 patients with NSCLC during the operations. Each lymph node sample was divided into two same parts in size. The one half part of lymph node should be examined by conventional histopathologic examination and immunohistochemical (IHC) staining. All the remaining lymph node samples of each patient should be mixed together for the reverse transcriptase-polymerase chain reaction (RT-PCR) if they located in the same region. As long as the presence of metastatic tumor in one lymph node was found by H&E staining, all other lymph node samples in the same region should not be detected by IHC staining or RT-PCR techniques. Frozen tissue sections of 135 lymph nodes that were staged as free of metastases by conventional histopathologic examination were screened by IHC staining. 31 lymph nodes showed single cell or cells clusters. Of 39 groups mixed regional lymph nodes which were diagnosed to be devoid of metastases by conventional histopathologic examination, 11 groups were found to have positive reactions to cytokeratin 19-mRNA by RT-PCR. There was a correlation between IHC staining and RT-PCR for detection of nodal micrometastases of NSCLC (U = 7.682, P = 0.0001). IHC staining analysis can facilitate the detection of occult micrometastases in lymph nodes of NSCLC, and its assessment of nodal micrometastases can provide a refinement of TNM stage for partial patients with stage I to II. RT-PCR has the same value as IHC staining in detection of lymph nodal micrometastases. RT-PCR technique can reduce expense of the detecting micrometastases in lymph nodes.

Effects of 17beta-estradiol and progesterone on transcription of human papillomavirus 16 E6/E7 oncogenes in CaSki and SiHa cell lines

Several in vitro studies have addressed the interactions between estrogen/progesterone and human papillomavirus (HPV), but the results are controversial. We evaluated the effects of estrogen and progesterone and their antagonists on messenger RNA expression of HPV16 E6/E7 in HPV16-positive cell lines CaSki and SiHa with real-time reverse-transciptase polymerase chain reaction method. Colorimetric assay with tetrazolium salt (WST-1) and flow cytometry were used for testing proliferation and apoptosis. No statistically significant changes were found after hormone treatment in the expression of HPV16 E6/E7 or hormone receptors in CaSki and SiHa cell lines. Progesterone increased cell proliferation in both the cells, while estrogen increased proliferation of SiHa cells only. Estrogen seemed to protect the CaSki cells from apoptosis, and tamoxifen did not abrogate this effect. Progesterone slightly increased apoptosis of CaSki cells, and this effect was neutralized with RU486. In this study, estrogen and progesterone did not change either the transcription levels of HPV16 E6/E7 or estrogen receptor or progesterone receptor levels. Hormone receptor antagonists had no effect on transcription. Both hormones might have a permissive effect for the growth of cervical cancer, by promoting cell proliferation and making the cells vulnerable to mutations. In addition, estrogen acts as an antiapoptotic agent allowing growth advance of the cells infected with oncogenic HPV.

Effects of 17β-estradiol and progesterone on transcription of human papillomavirus 16 E6/E7 oncogenes in CaSki and SiHa cell lines

Several in vitro studies have addressed the interactions between estrogen/progesterone and human papillomavirus (HPV), but the results are controversial. We evaluated the effects of estrogen and progesterone and their antagonists on messenger RNA expression of HPV16 E6/E7 in HPV16-positive cell lines CaSki and SiHa with real-time reverse-transciptase polymerase chain reaction method. Colorimetric assay with tetrazolium salt (WST-1) and flow cytometry were used for testing proliferation and apoptosis. No statistically significant changes were found after hormone treatment in the expression of HPV16 E6/E7 or hormone receptors in CaSki and SiHa cell lines. Progesterone increased cell proliferation in both the cells, while estrogen increased proliferation of SiHa cells only. Estrogen seemed to protect the CaSki cells from apoptosis, and tamoxifen did not abrogate this effect. Progesterone slightly increased apoptosis of CaSki cells, and this effect was neutralized with RU486. In this study, estrogen and progesterone did not change either the transcription levels of HPV16 E6/E7 or estrogen receptor or progesterone receptor levels. Hormone receptor antagonists had no effect on transcription. Both hormones might have a permissive effect for the growth of cervical cancer, by promoting cell proliferation and making the cells vulnerable to mutations. In addition, estrogen acts as an antiapoptotic agent allowing growth advance of the cells infected with oncogenic HPV.

Expression of augmenter of liver regeneration (ALR) in human liver cirrhosis and carcinoma

To determine the expression of a protein termed augmenter of liver regeneration (ALR), recently found to have a specific and beneficial effect on the process of liver regeneration in normal and diseased human liver. ALR expression in normal and cirrhotic human livers with various underlying diseases as well as in tissue samples of hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) was analysed by immunohistochemistry and quantitative reverse transciptase-polymerase chain reaction (RT-PCR). Expression analysis of ALR in total liver protein extracts by Western blotting showed mainly dimeric ALR protein. Immunohistochemically, cytosolic and perinuclear immunosignals were found in hepatocytes and cholangiocytes in normal, cirrhotic or cancerous liver tissue and only weak signals in some endothelial cells in normal livers. Quantitative mRNA analysis revealed significantly increased ALR expression in cirrhosis compared with normal liver tissue. In HCC and CCC ALR mRNA expression was also significantly enhanced compared with normal liver tissue, but expression levels did not differ from the matching non-neoplastic tissue in the same patient. The findings suggest an important role for ALR in hepatocellular regeneration in liver cirrhosis as well as in hepatocarcinogenesis and therefore its potential value in the clinical diagnosis of hepatic cirrhosis and cancer.

Expression of Cdx2 in Early GRCL of Barrett?s Esophagus Induced in Rats by Duodenal Reflux

The intestine-specific caudal-related homeobox transcription factor Cdx2 is widely accepted to play a key role in intestinal development and differentiation in mammals. We studied the role of Cdx2 in the development of Barrett's esophagus (BE). In previous studies, we have shown a sequence of morphological changes of squamous epithelium leading to BE, found a peculiar metaplastic change common to other parts of gut, and proposed the concept of a "gut regenerative cell lineage" (GRCL). The GRCL is characterized by pyloric-foveolar metaplasia with goblet cell metaplasia, which occurs in the regenerative process in response to chronic inflammation. BE very likely develops through the GRCL, and we studied the expression of Cdx2 in various lesions of rat esophageal mucosa induced by duodenal reflux, using reverse transciptase-polymerase chain reaction and immunohistochemistry against Cdx2. We found that Cdx2 was expressed not only in specialized columnar epithelium (SCE) of BE, but also in several pyloric gland and foveolar metaplastic cells which developed in the basal layer of the squamous epithelium at an earlier stage of SCE development. These findings indicate that Cdx2 plays a crucial role in directing intestinal-type differentiation of the GRCL.

Expression profiling of MMA-1a and splice variant MMA-1b: new cancer/testis antigens identified in human melanoma.

Using high-density oligonucleotide array analysis, we have recently compared the gene expression profiles of 2 human melanoma cell lines with marked difference in metastatic behavior after subcutaneous inoculation into nude mice (de Wit et al., Melanoma Res, in press). We identified an expressed sequence tag (EST), which we called malignant melanoma-associated 1 (MMA-1a), showing evident differential expression between the 2 cell lines. The MMA-1a gene is localized on chromosome 21q22.2 and its mRNA exists of 4 exons. Homology search displayed a splice variant of MMA-1a that lacks exon 3 and that was called MMA-1b. Expression profiles of MMA-1a and MMA-1b are determined by reverse transciptase polymerase chain reaction (RT-PCR) analysis. Among 30 different normal tissue samples, expression of MMA-1a and MMA-1b was exclusively found in the testis after a first PCR of 30 cycles. Even more sensitive screening achieved by performing multiple semi-nested RT-PCR showed no or very low expression in the other normal tissues tested. During melanocytic tumor progression, MMA-1a and/or MMA-1b exhibited an emergence of expression in primary melanoma (20%) and melanoma metastasis samples (30%) after only 1 round of PCR. Expression of MMA-1a and/or MMA-1b was also identified in other tumor cell lines and fresh tumor samples of variable origin, e.g., lung, liver, bladder and soft tissues (sarcomas). We conclude that MMA-1a and MMA-1b are new members of the family of cancer/testis antigens.

Abnormal E-cadherin expression and prostate cell blood dissemination as markers of biological recurrence in cancer

Until now, no molecular parameter has been available for predicting the metastatic potential of prostate tumours, which leaves their outcome uncertain despite an apparent benign histology or early stage. Abnormal expression of adhesion molecules, such as E-cadherin, can be contributing factors for increased invasiveness and metastatic potential. Histological analysis for E-cadherin expression was carried out on paraffin-embedded tumour tissues. Tumour metastatic potential was indirectly evaluated by detecting circulating prostate cells (CPC), using reverse transciptase-polymerase chain reaction (RT-PCR) and prostate-specific membrane antigen (PSMA) as a target. Patients were followed-up for a median of 14 months (range 10–19 months) after surgery with serum prostate-specific antigen (PSA) level measurement. Interestingly, 23 of 44 localised tumours exhibited aberrant E-cadherin expression. Prior to primary surgery, PSMA RT-PCR detected the spread of prostate cells to the blood in 24 patients. Statistical analysis showed that abnormal E-cadherin expression in the tumours was the only variable that was independently correlated with prostate cell dissemination in the blood ( P<0.0001). In logistic regression analysis, abnormal E-cadherin expression was a significant independent predictor for a later biological relapse. This impaired adhesion status was clearly correlated with a haematogenous spread of the primary tumour cells. It could therefore be an objective way to restrict the indications for radical surgery to patients not presenting with this feature.