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PubMedDate not supplied

Differential expression of prolactin receptor (PRLR) in normal and tumorous adrenal tissues: separation of cellular endocrine compartments by laser capture microdissection (LCM).

PRL stimulates adrenal steroidogenesis. In this study, we compared the PRLR expression in normal and tumorous adrenal tissues and investigated a potential proliferative effect of PRL in adrenal cells. mRNA expression of long and intermediate forms of PRLR was detected in both normal adrenal cortex as well as benign and malignant adrenal tumors and in the human adrenocortical carcinoma cell line NCI-H295. Molecular analysis of cells procured by LCM clearly demonstrated that PRLR mRNA is expressed in the adrenal cortex but not in the medulla. Immunostaining revealed PRLR protein in all three zones of the normal adrenal cortex. Furthermore, adrenal carcinomas and adenomas stained positive for the PRLR, while in phaeochromocytomas as in the normal adrenal medulla, no specific staining was observed. By WST-1 test, we could show that PRL (10(-7) M) decreased proliferation and viability of adrenal cells in primary cell culture suggesting that PRL is not a mitogenic factor of adrenocortical cells.

Adrenal Cortex↗
PubMed1997

c-myc gene abnormalities in mucosa-associated lymphoid tissue (MALT) lymphomas.

c-myc gene abnormalities associated with lymphomagenesis, including rearrangements and mutations in the regulatory region between exon I and intron I, have been studied in 54 MALT lymphomas (43 low and 11 high grade) and 36 nodal lymphomas (27 low and 9 high grade). By Southern blot analysis, none of the 54 MALT lymphomas but 2 of 36 nodal lymphomas had c-myc gene rearrangements. Defined tumour cell populations from all MALT lymphoma cases were isolated by microdissection from frozen tissue sections and analysed by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and direct sequencing for somatic mutations in the exon I/intron I region of the gene. Point mutations in this region were identified in nine cases of MALT lymphomas (7/43 = 16.2 per cent of low grade; 2/11 = 18.1 per cent of high grade). These mutations were located at either the exon I/intron I border of myc intron factor (MIF) binding sites, which are critical in the negative regulation of c-myc expression. Of the nodal lymphomas, only the two cases (5-6 per cent) with c-myc gene rearrangement showed scattered or clustered mutations. These results suggest that c-myc mutations in MALT lymphomas are unlikely to be associated with chromosome translocation, which is the main cause of somatic mutations observed in other types of lymphomas. The mutations involving the c-myc regulatory regions may play a pathogenetic role in at least a proportion of MALT lymphomas.

Base Sequence↗
PubMed1999

Loss of heterozygosity at 1p36 predicts poor prognosis in gastrointestinal stromal/smooth muscle tumors.

Gastrointestinal stromal/smooth muscle tumors are uncommon neoplasms for which current criteria for diagnosing malignancy (size and mitotic index) sometimes fail to predict outcome. Cytogenetic studies reveal frequent chromosome 1 abnormalities in these tumors, but significant underlying molecular changes have not been elucidated, and their significance is unknown. DNA was obtained from the formalin-fixed, paraffin-embedded tissue of 80 gastrointestinal stromal/smooth muscle tumors. Tumors were topographically microdissected from surrounding normal tissue; microsatellite markers from tumor and normal tissue were amplified by PCR in the regions of chromosome 1p36 (D1S199, D1S228, D1S450, D1S214, D1S243), 1p12 (D1S418),1p13 (D1S252, D1S514), and 1q32(D1S103). The presence or absence of heterozygosity for each case was mapped at each informative marker. Relationships among loss of heterozygosity (LOH), tumor size, mitotic index, and survival were investigated using correlation analysis, Kaplan-Meier plots, and the Cox model. LOH at 1p36 was found in 24 of 80 cases, suggesting the possibility of a tumor suppressor gene at 1 p36 near the site of a suspected neuroblastoma tumor suppressor gene. Patients whose tumors demonstrated LOH at 1 p36 had significantly shorter survival (p = 0.017) than those whose tumors did not. LOH at 1 p36 retained independent prognostic significance in a multivariate model that included KIT mutation status and tumor size; the mitotic index, however, did not retain independent significance in such a model. LOH was observed at 1 p12-1p13 (most frequently at 1p13.3) in 19 of 80 cases, but loss of heterozygosity at this site did not influence survival. No LOH was observed near 1q32. These findings provide strong evidence for a prognostically significant tumor suppressor gene in the region of chromosome 1p36.3.

Base Sequence↗
PubMed1984

Bioluminescence procedures for the measurement of NAD(P) dependent enzyme catalytic activities in submicrogram quantities of rabbit and human nephron structures.

Reduced flavin mononucleotide dependent luciferase (EC 1.14.14.3) from Photobacterium fischeri has been used to measure NAD(P) dependent enzymes in submicrogram quantities of tissue homogenates and isolated structures of rabbit and human kidney. The procedure for measuring NAD(P)H was optimized, with internal standardization, to give a linear constant signal between 1 and 100 pmol. This method was applied to the measurement of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) along the various structures of the rabbit nephron, microdissected from fresh tissue slices. Blank and recovery measurements were performed on each structure, and enzyme catalytic activities were calculated on the basis of tubular length and protein. Glucose-6-phosphate dehydrogenase was found to be present in all nephron structures with highest catalytic activities in glomeruli, thin limbs of Henle's loop and medullary collecting tubules. Lowest catalytic activities were detected in the pars recta of proximal tubules, the distal convoluted and the connecting tubule. The mitochondrial 3-hydroxybutyrate dehydrogenase exhibited a different distribution pattern: Highest catalytic activities were found in the cortical ascending limbs of Henle's loop, the proximal and distal convoluted tubule. An unusual internephron heterogeneity for this enzyme was found in the distal convoluted tubule. Catalytic activities in thin limbs of Henle's loop, the medullary ascending limb, and the cortical and medullary collecting tubule were not significantly different from blank activities. The results obtained in isolated nephron segments agreed with those calculated from cortex and medullary homogenates. In a preliminary experiment on human kidney it could be demonstrated that the procedures can be applied to fresh human biopsy samples. The bioluminescence method offers several advantages (simplicity, rapidity) in comparison with the classical techniques used for ultramicro analysis of tissue enzymes (enzymatic cycling, radiochemical tests). The results confirm the biochemical heterogeneity of nephron structures and allow conclusions about species dependent functional differences of the pentose-phosphate cycle and renal ketone body metabolism along the nephron.

Animals↗
PubMed1999

MicroSAGE: a modified procedure for serial analysis of gene expression in limited amounts of tissue.

Serial Analysis of Gene Expression (SAGE) is a powerful expression profiling method, allowing the analysis of the expression of thousands of transcripts simultaneously. A disadvantage of the method, however, is the relatively high amount of input RNA required. Consequently, SAGE cannot be used for the generation of expression profiles when RNA is limited, i.e. in small biological samples such as tissue biopsies or microdissected material. Here we describe a modification of SAGE, named microSAGE, which requires 500- to 5000-fold less starting material. Compared with SAGE, microSAGE is simplified due to incorporation of a 'single-tube' procedure for all steps from RNA isolation to tag release. Furthermore, a limited number of additional PCR cycles are performed. Using microSAGE gene expression profiles can be obtained from minute quantities of tissue such as a single hippocampal punch from a rat brain slice of 325 micrometers thickness, estimated to contain, at most, 10(5) cells. This method opens up a multitude of new possibilities for the application of SAGE, for example the characterization of expression profiles in tissue biopsies, tumor metastases or in other cases where tissue is scarce and the generation of region-specific expression profiles of complex heterogeneous tissues.

Animals↗
PubMed1999

Presence of simian virus 40 sequences in malignant mesotheliomas and mesothelial cell proliferations.

Malignant mesotheliomas (MMs) are pleural-, pericardial-, or peritoneal-based neoplasms usually associated with asbestos exposure. Mesothelial cells are biphasic and may give rise to epithelial and sarcomatous MMs. In addition, benign or atypical proliferations of mesothelial cells may occur in response to many stimuli. There have been recent reports of simian virus 40 (SV40) DNA large T antigen (Tag) sequences in pleural MMs. To further understand the relationship between SV40, MMs, and mesothelial proliferations, we studied 118 MMs from multiple sites in Germany and North America, including 93 epithelial pleural, 14 sarcomatous or mixed pleural MMs, and 11 peritoneal MMs. In 12 pleural MMs, adjacent noninvasive tumor foci were identified and studied separately. Information about asbestos exposure (detailed history and/or microscopic examination for asbestos bodies) was available from 43 German patients. In addition, 13 examples of reactive mesothelium and 20 lung cancers from the United States were tested. DNA was extracted from frozen tumor and adjacent nontumorous tissues or after microdissection of archival formalin-fixed, paraffin-embedded microslides. Two rounds of PCR were performed with primers SVFor 3 and SVRev, which amplify a 105 bp region specific for SV40 Tag. The specificity of the PCR product was confirmed in some cases by sequencing. Our major findings were: 1) Specific SV40 viral sequences were present in 57% of epithelial invasive MMs, of both pleural and peritoneal origin. No significant geographic differences were found, and frozen and paraffin-embedded tissues were equally suitable for analysis. 2) There was no apparent relationship between the presence of SV40 sequences and asbestos exposure. 3) SV40 sequences were present in the surface (noninvasive) components of epithelial MMs. 4) SV40 sequences were not detected in MMs of sarcomatous or mixed histologies. 5) Viral sequences were present in two of 13 samples (15%) of reactive mesothelium. 6) Lung cancers lacked SV40 sequences, as did non-malignant tissues adjacent to MMs. Our findings demonstrate the presence of SV40 sequences in epithelial MMs of pleural and peritoneal origin and their absence in tumors with a sarcomatous component. Viral sequences may be present in reactive and malignant mesothelial cells, but they are absent in adjacent tissues and lung cancers.

Antigens, Polyomavirus Transforming↗
PubMed2002

Detection of TP53 mutation in ameloblastoma by the use of a yeast functional assay.

BACKGROUND: Mutations in TP53 have been observed in a variety of tumors including oral lesions, though there are no reports in ameloblastomas. The purpose of the study was to examine the TP53 status of ameloblastomas using newly developed yeast functional assay whose accuracy and sensitivity has been proven to be higher than those of the previous DNA structure-based methods such as single strand conformation polymorphism (SSCP) analysis. METHODS: TP53 status was analyzed by yeast functional assay and DNA sequencing in 12 cases of ameloblastoma which were diagnosed histologically and represented the clinical features of a benign tumor. After the extraction of RNA from the frozen tissue samples without microdissection, reverse transcription (RT)-PCR was carried out and these samples were used. The assay can detect mutations of p53 mRNA between codons 67 and 347 by the DNA-binding activity of the protein and reveal them as red colonies. RESULTS: One case of 47-year-old male gave 17% red colonies of yeast and the other 11 cases gave 5.4% (mean; range, 3-8%). To confirm this result, we obtained other nine samples from the case of 17% red colonies, which contained or did not contain tumor tissues, and analyzed them by the assay. Seven samples that were histologically negative for tumor cells gave 4.7% red colonies (mean; range, 1-7%). Two samples that were histologically positive for tumor cells gave 20 or 46% of red colonies. The p53 plasmids were recovered from the red colonies of these three samples showing high red colony ratios and were subjected to sequencing analysis after purification. In all these samples, the same clonal mutation of TGT (Cys) 238 TAT (Tyr) was demonstrated. CONCLUSIONS: These results suggest that TP53 mutation may be involved in molecular pathogenesis in a subset of ameloblastomas, though it is infrequent.

Adolescent↗
PubMed2000

Molecular genetic analysis of archival gliomas using diagnostic smears.

Investigation of the clinical significance of genetic alterations in gliomas requires molecular genetic analysis using samples from retrospective or prospective clinical studies. However, diagnostic tissue is often severely limited and because of fixation, paraffin-embedded tissues (PET) contain degraded DNA. Intra-operative cytological preparations (smears) archived after diagnosis may represent an additional source of clinical material for genetic analysis. In this study, tissue samples were obtained by precision microdissection of archived diagnostic smears from 20 cases (1961-1999). All samples produced polymerase chain reaction (PCR) products for the beta globin gene, but the most recent samples amplified best and gave longer amplimers. For six cases, direct comparison was made between samples microdissected from smears and the corresponding PET. Samples from smears showed improved PCR performance and similar alleles on microsatellite marker analysis. One case, with smears of uninvolved cortex and tumour tissue available for microdissection, showed allelic imbalance at 10q23 on the basis of the smear results alone. PCR products from smears were shown to be suitable for direct sequence analysis (p53 gene). A PTEN mutation, found previously in an anaplastic astrocytoma by analysis of PET, was detected in the corresponding diagnostic smear. The results of this study indicate that tissue samples microdissected from diagnostic intra-operative cytological preparations may be suitable for molecular genetic analysis of gliomas.

Archives↗
PubMed2000

Influence of histochemical and immunohistochemical stains on polymerase chain reaction.

The polymerase chain reaction (PCR) analysis of DNA extracted from tissue sections can be applied to a variety of research and diagnostic protocols. To analyze selectively the specific areas of tissue, a direct microdissection of histochemically or immunohistochemically stained sections, if satisfactory for PCR, is helpful. However, the influence of various staining methods on PCR has been poorly investigated. In this study, paraffin sections of formalin-fixed lymph node samples were histochemically stained with Mayer's hematoxylin, eosin Y, methyl green, or May-Grunwald solution and immunostained for CD45 using 3,3'-diaminobenzidine (DAB), DAB with cobalt ion (DAB-Co), or new fuchsin as the chromogen. In addition, unstained sections were treated with trypsin, microwave, or pressure cooker, the techniques frequently used in immunostains for antigen unmasking. DNA was extracted from each section, and the PCR efficiency in amplifying a 110 bp portion of the beta-globin gene was evaluated by two parameters: the cycle count in which the first visible band was obtained (CYCLE(min)) and the maximum amount of PCR products (CONC(max)). The hematoxylin stain showed a significantly prolonged CYCLE(min) (P < .01) and lower CONC(max) (P < .05) in comparison with unstained and untreated control sections. The May-Grunwald stain showed a prolonged CYCLE(min) (P < .01), although the CONC(max) was not significantly different from that of the control (P = .051). The eosin and methyl green stains showed no effects against PCR. In immunostains, the DAB-Co method showed a lower CONC(max) (P < .05), whereas the CYCLE(min) was not prolonged. The DAB and new fuchsin methods had no untoward effects. Antigen-unmasking treatments showed deteriorating effects on PCR. The trypsin treatment significantly prolonged the CYCLE(min) (P < .01), and the PCR amplification did not reach the "plateau" level with a maximum of 60 cycles. The PCR efficiency was worse in microwave or pressure cooker treatment, with neither CYCLE(min) nor CONC(max) being obtained. When target areas from sections for subsequent PCR amplification are microdissected, methyl green is most suitable as a dye for nuclear staining. The immunohistochemical visualization with DAB or new fuchsin yields no unfavorable effects. A successful PCR amplification may not be expected in sections that are pretreated in a microwave oven or pressure cooker.

Beta-Globulins↗
PubMed2004

Histo-blood group ABO antigen in oral potentially malignant lesions and squamous cell carcinoma--genotypic and phenotypic characterization.

Loss of histo-blood group A/B antigens is frequent in oral cancer. It is unclear whether this alteration is due to loss of the chromosomal region encoding the genes. The aim was to investigate genotypic alterations in the ABO locus in oral potentially malignant lesions and carcinomas. Seventy-three cases which expressed A/B antigen in normal epithelium by immunohistochemical (IHC) staining were investigated. Both tumour and normal cells were collected from paraffin-embedded tissue by laser microdissection. DNA was extracted and analysed by PCR coupled with restricted digestion analysis in order to establish the ABO genotype. Total and patchy loss of A/B antigen expression was found in 24/32 carcinomas, 6/7 leukoplakias with severe dysplasia, 12/17 leukoplakias with mild and moderate dysplasia, and 6/17 leukoplakias without dysplasia. Specific A/B allele loss was found in 8/24 cases with carcinoma and 3/24 cases with mild and moderate dysplasia by genotyping analysis. O allele loss was found in 10 cases involving all four groups. In patients with heterozygous genotypes, A/B allelic loss by genotyping analysis was always followed by loss of A/B antigen expression by IHC staining. Loss of A/B antigen expression in tissues which had intact ABO alleles was, however, found and may be explained by other genetic and epigenetic changes.

ABO Blood-Group System↗
PubMed2000

Reproductive tissue renin gene expression in preeclampsia.

OBJECTIVE: Using an analogy with renin gene overexpression, low-renin hypertension animal models, we wished to test the hypothesis that renin gene expression is increased in decidua basalis in human gestation with preeclampsia. METHODS: Human placentas were obtained immediately after delivery from 11 control (C) and 11 preeclamptics (PE). Tissue samples were microdissected and renin gene expression in decidua basalis (DB), chorionic villi (CV), and decidua vera (DV) was measured using dot-blot hybridization. RESULTS: Overall renin gene expression is highest in decidua basalis (mean +/- SEM, 2.66 +/- 0.69 densitometry area units) compared to chorionic villi (mean +/- SEM, 1.85 +/- 0.5) or compared to decidua vera (mean +/- SEM, 1.63 +/- 0.9) (both t-tests p = 0.001 two-tailed and analysis of variance p = 0.0001). Renin gene expression in DB and in CV was similar in both preeclamptic and normal pregnancies (DB mean +/- SEM C 2.79 +/- 0.96 versus PE 2.54 +/- 1.04, and CV mean +/- SEM C 2.11 +/- 0.91 versus PE, 1.59 +/- 0. 44). Renin gene expression in DV was approximately threefold higher in tissues from preeclamptics compared to control (mean +/- SEM PE 2. 44 +/- 1.76 versus C 0.82 +/- 0.42). Using the median value of 0.5 units for DV as cutoff, the preeclamptics displayed higher renin gene expression (chi square p = 0.033, two tailed). CONCLUSION: Our data suggest that renin gene expression is increased in preeclampsia in decidua vera. This may explain previously reported increased renin secretion in uterine circulation in preeclampsia.

Adult↗
PubMed2005

Multitargeted effects of statin-enhanced thrombolytic therapy for stroke with recombinant human tissue-type plasminogen activator in the rat.

BACKGROUND: Microvascular dysfunction posttreatment of stroke with recombinant human tissue-type plasminogen activator (rht-PA) constrains the therapeutic window to 3 hours. Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) promote vascular thrombolysis and reduce the inflammation response. We therefore investigated the neuroprotective effects of a combination of atorvastatin and delayed rht-PA treatment in a rat model of embolic stroke. METHODS AND RESULTS: Rats subjected to embolic middle cerebral artery occlusion were treated with atorvastatin in combination with rht-PA 4 hours after stroke. Magnetic resonance imaging measurements revealed that combination treatment with atorvastatin and rht-PA blocked the expansion of the ischemic lesion, which improved neurological function compared with saline-treated rats. Real-time reverse transcription-polymerase chain reaction analysis of single endothelial cells isolated by laser-capture microdissection from brain tissue and immunostaining showed that combination treatment downregulated expression of tissue factor, von Willebrand factor, protease-activated receptor-1, intercellular adhesion molecule-1, and matrix metalloproteinase-9, which concomitantly reduced cerebral microvascular thrombosis and enhanced microvascular integrity. Combination treatment did not increase cerebrovascular endothelial nitric oxide synthase (eNOS) levels or eNOS activity, and inhibition of NOS activity with N-nitro-L-arginine methyl ester did not block the beneficial effects of combination treatment on stroke. Furthermore, combination treatment compared with thrombolytic monotherapy increased cerebral blood flow and reduced infarct volume in eNOS-null mice. CONCLUSIONS: These data demonstrate that combination treatment with atorvastatin and rht-PA exerts a neuroprotective effect when administered 4 hours after stroke and that the therapeutic benefits are likely attributed to its multitargeted effects on cerebrovascular patency and integrity.

Animals↗
PubMed2000

Diagnostic primer sets for microsatellite instability optimized for a minimal amount of damaged DNA from colorectal tissue samples.

BACKGROUND: The diagnosis of microsatellite instability from a minimal amount of highly damaged DNA, extracted from formalin-fixed, paraffin-embedded tissue by the microdissection method, is difficult. Therefore, optimized primer sets were newly designed for substitution of documented ones. METHODS: DNA was extracted from 15 archival colorectal carcinomas and used as templates for polymerase chain reaction. Nine standard microsatellite markers (BAT-25, BAT-26, BAT-40, D18S69, D2S123, D5S346, D10S197, D17S250, and D18S58) were selected for diagnosis of microsatellite instability in colorectal carcinomas. All polymerase chain reaction conditions for primer sets were unified to save experimental time. RESULTS: The primer sets for the latter five markers documented in the literature were redesigned because of poor efficiency for damaged DNA. As a result, the number of DNA samples, sufficiently amplified at all markers, improved from 0% to 93%. CONCLUSIONS: Diagnostic primer sets for microsatellite instability, optimized for a minimal amount of damaged DNA from colorectal tissue samples, were established.

Colorectal Neoplasms↗
PubMed1995

Blood supply of subcutaneous tissue in the leg and its clinical application.

The vascular anatomy of the subcutaneous or adipose layer of the leg is described. The major arteries of the distal thigh and leg were injected with either a colored latex or an India ink and gelatin mixture to demonstrate the principal sources of blood supply to the skin and the superficial tissues of the leg. Microdissection and the technique of Spalteholz for dehydrating and clearing the tissues (rendering them transparent) facilitated the study of the direct and indirect arterial branches which form the three principal networks: the deep fascia, the subcutaneous or adipose layer, and the skin or the dermoepidermic layer. The results are of practical importance since the fascio-subcutaneous tissue layer can be used as a flap for covering soft tissue defects of the leg.

Adipose Tissue↗
PubMedDate not supplied

Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling.

The goal of this study was the development of a method for quantitative expression proteomics on the limited sample amounts obtained through laser capture microdissection (LCM) of tissues, e.g., approximately 10 000 cells, which typically contain roughly 1-4 microg protein. The 16O/18O labeling method was selected as an approach to measure differential expression. A sample preparation protocol including lysis, digestion and 16O/18O labeling was first developed for LCM cell samples. The selected protocol was examined using two LCM caps of 10 000 cells from invasive ductal carcinoma of the breast and shown to be repeatable. A further test of LC-IT-MS/MS in combination with the 16O/18O post-digestion labeling method for studying low level samples was conducted first on a single protein (BSA) and then on a 5-standard protein mixture digest of different protein amounts, each with a total content approximately 1 microg. Next, protein expression was compared between 10 000 cells, each of microdissected normal ductal epithelium and metastatic ductal carcinoma, using the developed method. The proteins from the microdissected cells were extracted, precipitated, digested with trypsin and then 16O/18O labeled. The normal and metastatic cell samples were analyzed using reversed phase LC-ESI-MS/MS on the ion trap mass spectrometer. A total of 76 proteins were identified. Some, such as mitochondrial isocitrate dehydrogenase, actin and 14-3-3 protein xi/delta were found to be significantly up-regulated in the breast tumor cells.

Amino Acid Sequence↗
PubMed2006

Treatment of embolic stroke in rats with bortezomib and recombinant human tissue plasminogen activator.

Stroke elicits a progressive vascular dysfunction, which contributes to the evolution of brain injury. Thrombolysis with tissue plasminogen activator (tPA) promotes adverse vascular events that limit the therapeutic window of stroke to three hours. Proteasome inhibitors reduce vascular thrombotic and inflammatory events, and consequently protect vascular function. The present study evaluated the neuroprotective effect of bortezomib, a potent and selective inhibitor of the proteasome, alone and in combination with delayed thrombolytic therapy on a rat model of embolic focal cerebral ischemia. Treatment with bortezomib reduces adverse cerebrovascular events including secondary thrombosis, inflammatory responses, and blood brain barrier (BBB) disruption, and hence reduces infarct volume and neurological functional deficit when administrated within 4 h after stroke onset. Combination of bortezomib and tPA extends the thrombolytic window for stroke to 6 h, which is associated with the improvement of vascular patency and integrity. Real time RT-PCR of endothelial cells isolated by laser-capture microdissection from brain tissue and Western blot analysis showed that bortezomib upregulates endothelial nitric oxide synthase (eNOS) expression and blocks NF-kappaB activation. These results demonstrate that bortezomib promotes eNOS dependent vascular protection, and reduces NF-kappaB dependent vascular disruption, all of which may contribute to neuroprotection after stroke.

Animals↗
PubMed2005

A novel component of the ubiquitin pathway, ubiquitin carboxyl extension protein 1 is overexpressed in prostate cancer.

Prostate cancer is among the most common tumors in industrialized nations. However, little is known about the molecular events underlying its development. In the present study we used suppressive subtraction hybridization (SSH) in combination with laser-assisted microdissection in order to compare gene expression between prostate carcinoma and the normal prostate proper. Both are mixed tissues which consist of an epithelial and a stromal compartment. We first compared mRNA (cDNA) expression by SSH and then used real-time quantitative RT-PCR analysis of microdissected tissue probes in order to verify differential expression of subtracted cDNA clones. We also used differentially expressed cDNAs for the synthesis of radiolabelled riboprobes in order to attribute differential expression to specific cell types in tissue sections by in situ hybridization. Using this approach we found an up-regulation of ubiquitin carboxyl extension protein 1 (UBCEP-1) mRNA in prostate carcinoma cells compared to the normal glandular epithelium of the prostate proper. UBCEP-1 mediated ubiquitin chain elongation may promote prostate carcinoma development by increasing via the proteasome pathway the degradation of proteins which are involved in growth inhibition or apoptosis.

Aged↗
PubMed2006

Laser-assisted microdissection for real-time PCR sample preparation.

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of bio-molecules from microdissected tissue samples have been analysed.

Cell Line↗