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PubMed2005

Real-time polymerase chain reaction and laser capture microdissection for the diagnosis of BK virus infection in renal allografts.

We used real-time polymerase chain reaction (PCR) technology to detect BK virus (BKV) in H and E-stained kidney biopsy sections, using laser capture microdissection. Renal allograft biopsy specimens from 4 patients with the histopathologic diagnosis of BKV-associated nephropathy (BKVAN; group 1) and 3 patients suspected to have BKVAN but without diagnostic histologic features (group 2) were retrieved. Diagnostic inclusion-bearing cells were microdissected by laser capture microscopy from group 1. Renal tubular epithelial cells were microdissected randomly in group 2. DNA was extracted and real-time amplification performed using primers targeting the large "T" and small "t" regions of the BKV and JC virus genomes. Tubular epithelial cells from a case without evidence of BKV infection were used as negative controls in a similar reaction. BKV presence was demonstrated only in epithelial cells containing typical viral inclusions. Group 2 and negative control samples were confirmed as negative for BKVAN. Real-time PCR technology can be used to detect BKV in H and E-stained, paraffin-embedded tissue sections. This technique detected BKV in tubular epithelial cells of renal allografts. To our knowledge, this is the first report of detecting BKV in laser capture microdissected renal biopsy specimens using real-time PCR.

BK Virus↗

PubMed1999

Composite low grade B-cell lymphomas with two immunophenotypically distinct cell populations are true biclonal lymphomas. A molecular analysis using laser capture microdissection.

Low grade B-cell lymphomas comprise several well defined, clinically and immunophenotypically distinct disease entities. Composite lymphomas showing phenotypic characteristics of more than one of these tumor subtypes in the same site are rare, and both common and separate clonal origins of the two tumor parts have been reported for cases studied by molecular methods. We describe the detailed immunohistochemical and molecular findings in three cases with features of composite low grade B-cell non-Hodgkin's lymphoma (B-NHL). All three neoplasms contained morphologically distinct but interwoven compartments of different cell types, which exhibited discordant expression of several markers, including CD5, CD10, CD43, and cyclin D1. According to their morphology and phenotypes, they were classified as mantle cell lymphoma and follicular lymphoma (Case 1), follicular lymphoma and small lymphocytic lymphoma (Case 2), and mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma (Case 3). PCR analysis of DNA obtained from whole tissue sections failed to reveal evidence for biclonality in any of the cases. We therefore isolated cell populations with different antigen expression patterns by laser capture microdissection and analyzed them by polymerase chain reaction amplification and sequencing of clonal immunoglobulin heavy chain gene rearrangements and oncogene rearrangements. Sequence analysis revealed unrelated clonal rearrangements in each of the two tumor parts in all three cases, suggesting distinct clonal origins. In addition, Case 1 showed a bcl-2 rearrangement present only in the follicular lymphoma part. Our findings suggest that low grade B-NHL with two distinct morphological and immunophenotypic patterns in the same anatomical site are frequently biclonal. This is in keeping with current classification schemes, which recognize subtypes of low grade B-NHL as separate disease entities. Furthermore, our analysis demonstrates the power of laser capture microdissection in revealing molecular microheterogeneity in complex neoplasms.

Aged↗

PubMed1993

Human glomeruli express TIMP-1 mRNA and TIMP-2 protein and mRNA.

Alterations in the balance between synthesis and degradation of extracellular matrix may result in glomerulosclerosis. The interaction between metalloproteinases and their inhibitors presumably modulates the rate of glomerular matrix degradation. We examined the gene expression of tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in human glomeruli and TIMP-2 protein in tissue sections. Kidney tissue was obtained from adults undergoing nephrectomy for renal tumor (n = 9) or biopsy for nephrosis and renal failure (n = 1). Glomeruli were microdissected and subjected to reverse transcription. TIMP cDNAs were quantitated by competitive polymerase chain reaction assays. Five nephrectomy specimens had normal glomeruli and four had diffuse glomerulosclerosis. TIMP-1 and TIMP-2 cDNA levels, detected in glomeruli from all patients, were increased fourfold and threefold, respectively, in patients with glomerulosclerosis. The elevated TIMP cDNA levels could not be attributed to an increased number of glomerular cells. TIMP-2 protein was detected within normal and sclerotic glomeruli. In conclusion, both TIMP genes were expressed in normal glomeruli, and their level of expression was increased in glomerulosclerosis associated with renal carcinoma, suggesting that expression of these inhibitors may correlate with the development of sclerosis.

Adult↗

PubMed1998

Isolation of genes identified in mouse renal proximal tubule by comparing different gene expression profiles.

An expression profile is a list based on a large scale sequencing of 1000 cDNA clones, showing the expressed genes and the abundance of their transcripts in a given cell or tissue (Okubo K et al: Nature Genet 2:173, 1992). We constructed an expression profile of mouse renal proximal tubules (PT) carefully isolated by microdissection in order to characterize its gene expression. Altogether 1000 clones were analyzed; there were 646 types of transcripts in PT, among which 196 were identical or homologous to the previously reported genes. The most abundant transcript was kidney-androgen regulated protein. By comparing the expression profile of PT with those obtained from other sources, several genes were identified only in PT. They included known transcripts and transcripts that were not homologous to the known genes. Three (GS4001, 3991, and 4059) of the non-homologous genes were analyzed by Northern blotting and in situ hybridization, and GS4001 and 4059 were predominantly expressed in the kidney, whereas GS3991 was detected in the liver as well as in the kidney. The sequence analysis of the full-size cDNAs demonstrated that GS4001 was a new member of aspartic proteinases and GS4059 was a novel gene. It also revealed that GS3991 was a mouse homologue of SA gene known to be expressed in PT. The expression profile of mouse PT and its comparison with those of other tissues and cells provide an alternate way of isolating genes predominantly expressed in PT, and also provides probes to study the molecular mechanisms of gene expression in the kidney.

3T3 Cells↗

PubMed1997

[Clonal analysis in cells using PCR and laser microdissection].

Clonality represents one of the hallmarks of neoplastic cell growth. X-chromosomal inactivation patterns have been used to determine clonality in various tumors. This approach is limited by admixture of polyclonal non-tumor stroma cells among which the monoclonal proliferation may be missed. In order to overcome this limitation, we combined a sensitive PCR based DNA analysis with a highly selective microdissection technique using a laser beam. In sections of intraductal mammary carcinomas tumor cell complexes of at least 100 cells were isolated by removing the surrounding stroma by laser irradiation. Thereby, tumor cells could be isolated without contaminating non-neoplastic elements. Clonality in these cells was determined using two X-chromosomal polymorphic sites-phosphoglycerate kinase 1 (PGK1) and human androgen receptor (HUMARA). Control experiments could show the polyclonal nature of the surrounding tissue. Moreover, complete destruction of DNA by laser irradiation was assured. The technique requires a certain amount of cells and DNA in order to avoid artefacts that result from preferential amplification of exclusively one X-chromosomal allele in small samples. We conclude that combination of laser-microdissection with PCR analysis of X-chromosomal inactivation patterns enables the detection of clonal cell populations in heterogeneous tissues. Studies of clonality in borderline cases between reactive and neoplastic proliferations or premalignant lesions are made possible by this technique.

Breast Neoplasms↗

PubMed2005

cDNA microarray analysis after laser microdissection in proliferating islets of partially pancreatectomized mice.

With islet transplantation having grown in popularity since the introduction of the Edmonton protocol, how to secure an unlimited source of islets has become an urgent problem. To resolve this problem, techniques to induce or proliferate islets are urgently required. To achieve this goal, gene expression analysis using a cDNA microarray in islets of partially pancreatectomized mice, in which the remaining islets regenerate and proliferate with insulin secretion and glucose responsiveness, provides us with valuable information. However, those experiments have two critical problems: first, how to selectively collect the regenerating or proliferating islets, and second, the shortage of total RNA extracted from one islet for a microarray analysis. A useful system was thus designed which combined laser microdissection, cDNA amplification by SMART PCR, which can maintain the relative expression profile of transcripts throughout reactions, and a cDNA microarray. Furthermore, this system is expected to contribute to future studies regarding not only islet regeneration but also the function of the islet itself, and this system may also be applicable to many other types of endocrine tissue. In this review, the details of this system are presented and discussed.

Animals↗

PubMed2001

Primary gene structure and expression studies of rodent paracellin-1.

The novel member of the claudin multigene family, paracellin-1/claudin-16, encoded by the gene PCLN1, is a renal tight junction protein that is involved in the paracellular transport of magnesium and calcium in the thick ascending limb of Henle's loop. Mutations in human PCLN1 are associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis, an autosomal recessive disease that is characterized by severe renal magnesium and calcium loss. The complete coding sequences of mouse and rat Pcln1 and the murine genomic structure are here presented. Full-length cDNAs are 939 and 1514 bp in length in mouse and rat, respectively, encoding a putative open-reading frame of 235 amino acids in both species with 99% identity. Exon-intron analysis of the human and mouse genes revealed a 100% homology of coding exon lengths and splice-site loci. By radiation hybrid mapping, the murine Pcln1 gene was assigned directly to marker D16Mit133 on mouse chromosome 16 (syntenic to a locus on human chromosome 3q27, which harbors the human PCLN1 gene). Mouse multiple-tissue Northern blot showed Pcln1 expression exclusively in the kidney. The expression profile along the nephron was analyzed by reverse transcriptase-PCR on microdissected nephron segments and immunohistochemistry of rat kidney. Paracellin-1 expression was restricted to distal tubular segments including the thick ascending limb of Henle's loop, the distal tubule, and the collecting duct. The identification and characterization of the rodent Pcln1 genes provide the basis for further studies of paracellin-1 function in suitable animal models.

Amino Acid Sequence↗

PubMed2006

Advanced techniques in placental biology -- workshop report.

Major advances in placental biology have been realized as new technologies have been developed and existing methods have been refined in many areas of biological research. Classical anatomy and whole-organ physiology tools once used to analyze placental structure and function have been supplanted by more sophisticated techniques adapted from molecular biology, proteomics, and computational biology and bioinformatics. In addition, significant refinements in morphological study of the placenta and its constituent cell types have improved our ability to assess form and function in highly integrated manner. To offer an overview of modern technologies used by investigators to study the placenta, this workshop: Advanced techniques in placental biology, assembled experts who discussed fundamental principles and real time examples of four separate methodologies. Y. Sadovsky presented the principles of microRNA function as an endogenous mechanism of gene regulation. J. Robinson demonstrated the utility of correlative microscopy in which light-level and transmission electron microscopy are combined to provide cellular and subcellular views of placental cells. A. Croy provided a lecture on the use of microdissection techniques which are invaluable for isolating very small subsets of cell types for molecular analysis. Finally, G. Rice presented an overview methods on profiling of complex protein mixtures within tissue and/or fluid samples that, when refined, will offer databases that will underpin a systems approach to modern trophoblast biology.

Animals↗

PubMed2004

Loss of the tumor suppressor PML in human cancers of multiple histologic origins.

BACKGROUND: The PML gene is fused to the RARalpha gene in the vast majority of acute promyelocytic leukemias (APL) and has been implicated in the control of key tumor-suppressive pathways. However, its role in the pathogenesis of human cancers other than APL is still unclear. We therefore assessed the status and expression of the PML gene in solid tumors of multiple histologic origins. METHODS: We created tumor tissue microarrays (TTMs) with samples from patients with colon adenocarcinoma (n = 109), lung carcinoma (n = 19), prostate adenocarcinoma (n = 36), breast carcinoma (n = 38), central nervous system (CNS) tumors (n = 51), germ cell tumors (n = 60), thyroid carcinoma (n = 32), adrenal cortical carcinoma (n = 12), and non-Hodgkin's lymphoma (n = 251) and from normal tissue corresponding to each histotype and analyzed PML protein and mRNA expression by immunohistochemistry and in situ hybridization, respectively. Tumor cell lines (n = 64) of various histologic origins were analyzed for PML protein and mRNA expression by immunofluorescence and northern blotting, respectively. DNA from microdissected tumor samples and cell lines was analyzed for PML mutations and loss of heterozygosity (LOH). For some tumor types, the association between PML expression and tumor stage and grade was analyzed. Statistical tests were two-sided. RESULTS: All normal tissues expressed PML protein. PML protein expression was reduced or abolished in prostate adenocarcinomas (63% [95% confidence interval [CI] = 48% to 78%] and 28% [95% CI = 13% to 43%], respectively), colon adenocarcinomas (31% [95% CI = 22% to 40%] and 17% [95% CI = 10% to 24%]), breast carcinomas (21% [95% CI = 8% to 34%] and 31% [95% CI = 16% to 46%]), lung carcinomas (36% [95% CI = 15% to 57%] and 21% [95% = 3% to 39%]), lymphomas (14% [95% CI = 10% to 18%] and 69% [95% CI = 63% to 75%]), CNS tumors (24% [95% CI = 13% to 35%] and 49% [95% CI = 36% to 62%]), and germ cell tumors (36% [95% CI = 24% to 48%] and 48% [95% CI = 36% to 60%]) but not in thyroid or adrenal carcinomas. Loss of PML protein expression was associated with tumor progression in prostate cancer (the progression from prostatic intraepithelial neoplasia to invasive carcinoma was associated with complete PML loss; P<.001), breast cancer (complete PML loss was associated with lymph node metastasis; P =.01), and CNS tumors (complete PML loss was associated with high-grade tumors; P =.003). PML mRNA was expressed in all tumor and cell line samples. The PML gene was rarely mutated and was not subject to LOH. CONCLUSIONS: PML protein expression is frequently lost in human cancers of various histologic origins, and its loss associates with tumor grade and progression in some tumor histotypes.

Adenocarcinoma↗

PubMed2006

[Application of laser capture microdissection in plant genomic research].

To a large degree, the growth and development of plants depend on the organ and/or cell type-specific expression of genes. However, it can be difficult to obtain sufficient number of specific cells from any developmental stage to analyze expression of genes. Laser capture microdissection (LCM) is a novel technique that allows us to collect pure targeted cell subgroup or even a single cell quickly and precisely, thus the problem of tissue heterogeneity in molecular analysis can be resolved successfully. In this paper, the principles of LCM were introduced and the application of LCM in plant was summarized in gene expression analysis, proteomics and plant-microbe interaction. Meanwhile, possible directions of LCM in plant were put forward.

Genome, Plant↗

PubMed2026

LCM-Enriched Proteomic Characterization of Antibody-Mediated Glomerular Damage and Complement Activation in Pre-Clinical Models.

Biologics, lipid nanoparticles, and other therapeutic modalities can result in adverse events, often detected as lesions during preclinical pathology assessments. Characterization of these lesions provides valuable information during drug development to contextualize mechanisms of injury and assess species translatability. Here, we investigated the utility of a laser capture microdissection (LCM)-enriched mass spectrometry proteomics approach to analyze two well-characterized preclinical models of regional (glomerular) injury: Passive Heyman Nephritis in rats and bovine gamma globulin-induced glomerular injury in nonhuman primates (NHPs). Using LCM-enriched proteomics, glomeruli were isolated from formalin-fixed paraffin-embedded kidney tissue in the rat model, enabling identification of 4,661 proteins and quantification of 3,410. Proteinuria measurements were compared with digital pathology metrics of glomerular morphology and proteomics results, with all modalities yielding concordant evidence of glomerular injury and proteomics confirming the role of complement activation. The same LCM- enriched proteomics workflow was applied to an NHP model of induced glomerular damage, identifying 4,623 proteins, quantifying 3,000, and confirming qualitative concordance with established features of complement-mediated glomerular injury. Together, these findings illustrate the applicability of LCM-enriched proteomics for region-specific characterization of antibody-mediated tissue injury and support its use as a hypothesis-generating platform in translational toxicologic pathology.

Animals↗

PubMed1999

Loss of heterozygosity at 3p in benign lesions preceding invasive breast cancer.

BACKGROUND: Loss of heterozygosity (LOH) at chromosome 3p is one of the most common genetic abnormalities identified in human cancers and has occasionally been noted in benign proliferative lesions predisposing to breast cancer. If the frequency of LOH at 3p in benign proliferative lesions correlates with the subsequent development of breast cancer, it may be possible to develop powerful tools for molecular risk assessment based on this technology. MATERIALS AND METHODS: Archival paraffin-embedded tissues from benign breast biopsies in five women who have developed breast cancer and three women who have not developed breast cancer were microdissected and allelotyped at 3p using six microsatellite markers. RESULTS: No LOH was detected in the biopsies from women who have not developed breast cancer. For women developing breast cancer, the proportion of informative loci showing LOH in the benign proliferative lesions was 0.47 as compared to 0.57 for the associated breast cancers. There was no LOH detected in epithelial DNA from a fibroadenoma. Of 15 informative loci, 4 (27%) showed LOH in both the benign proliferative lesion and the associated cancer; however, the actual parental allele lost was different in three of these four cases. CONCLUSIONS: These results suggest that there are specific patterns of genetic instability common to preneoplastic lesions and the breast cancers that subsequently develop even when the paired lesions are not clonally related. LOH analysis of benign breast epithelium may provide a tool for molecular risk assessment and a surrogate endpoint for breast cancer chemoprevention trials.

Adult↗

PubMed1998

Tissue sources of cytochrome P450 4A and 20-HETE synthesis in rabbit lungs.

We previously reported that 20-hydroxyeicosatetraenoic acid (20-HETE) is an endogenous cytochrome P450 (cP450) 4A metabolite of arachidonic acid (AA) in human lung tissue, and is a potent cyclooxygenase-dependent vasodilator of isolated pulmonary arteries. In the present investigations, we identified sources of cP450 4A immunospecific protein, messenger RNA (mRNA), and 20-HETE synthesis in rabbit lungs. Microsomes of peripheral lung tissue, airways, small and large vessels, and lysates of alveolar macrophages all express proteins of approximately 50 kD which cross-reacted with a primary antibody raised against rat liver cP450 4A1. Peripheral lung tissue, small and large pulmonary arteries, airways, and isolated vascular smooth muscle cells from small pulmonary arteries produced 20-HETE when incubated with AA. Expression of cP450 4A6/4A7 mRNA was readily detectable by reverse transcription-polymerase chain reaction using isoform-specific probes and 5 microg total RNA extracted from microdissected small pulmonary arteries. These data demonstrate that small pulmonary arteries express cP450 4A proteins and vascular smooth muscle cells derived from these arteries synthesize 20-HETE. Furthermore, cP450 4A appears to be widely distributed in rabbit tissue, raising the possibility that 20-HETE generated from nonvascular tissue could serve as a paracrine factor in the pulmonary circulation.

Animals↗

PubMed2005

Gene expression profiling of gastric cancer by microarray combined with laser capture microdissection.

AIM: To examine the gene expression profile of gastric cancer (GC) by combination of laser capture microdissection (LCM) and microarray and to correlate the profiling with histological subtypes. METHODS: Using LCM, pure cancer cells were procured from 45 cancerous tissues. After procurement of about 5000 cells, total RNA was extracted and the quality of RNA was determined before further amplification and hybridization. One microgram of amplified RNA was converted to cDNA and hybridized to cDNA microarray. RESULTS: Among 45 cases, only 21 were qualified for their RNAs. A total of 62 arrays were performed. These included 42 arrays for cancer (21 cases with dye-swab duplication) and 20 arrays for non-tumorous cells (10 cases with dye-swab duplication) with universal reference. Analyzed data showed 504 genes were differentially expressed and could distinguish cancerous and non-cancerous groups with more than 99% accuracy. Of the 504 genes, trefoil factors 1, 2, and 3 were in the list and their expression patterns were consistent with previous reports. Immunohistochemical staining of trefoil factor 1 was also consistent with the array data. Analyses of the tumor group with these 504 genes showed that there were 3 subgroups of GC that did not correspond to any current classification system, including Lauren's classification. CONCLUSION: By using LCM, linear amplification of RNA, and cDNA microarray, we have identified a panel of genes that have the power to discriminate between GC and non-cancer groups. The new molecular classification and the identified novel genes in gastric carcinogenesis deserve further investigations to elucidate their clinicopathological significance.

Gene Expression Profiling↗

PubMed1994

Expression of gelatinase A, a mediator of extracellular matrix remodeling, by tracheal gland serous cells in culture and in vivo.

Tracheal gland morphogenesis and gland hypertrophy in disease involve the penetration of epithelial cells into the submucosa, a process that requires digestion of the basal lamina and the surrounding extracellular matrix. We observed that bovine tracheal gland cells invaded collagen substrates and were inhibited from doing so in the presence of a metalloproteinase inhibitor GM6001. The gland cells, but not bovine tracheal surface epithelial cells, secreted a 72-kDa metalloproteinase. The purified enzyme could be activated with 4-aminophenylmercuric acetate and converted to an active 65-kDa form that was far more effective in degrading denatured collagen (gelatin) than nondenatured type I and IV collagens and was ineffective in degrading intact interstitial collagen fibers. At 25 degrees C, the initial rate of degradation of acid-solubilized type I collagen was approximately 50 mg of type I collagen cleaved per min per mg of enzyme, whereas acid-solubilized type IV collagen was degraded at approximately 250 mg cleaved per min per mg of enzyme. In contrast, at the same temperature, heat-denatured type I collagen was degraded 1000-fold more rapidly, while heat-denatured type IV collagen was cleaved 50-fold more rapidly. The activity of the enzyme was maximal at pH 7-8 and was completely abolished by the metalloproteinase inhibitors EDTA and 1,10-phenanthroline. In immunoblots, the enzyme was recognized by an antibody directed against human gelatinase A, the 72-kDa gelatinase. The purified enzyme disrupted the distribution pattern of type IV collagen in the gland basal lamina, as well as of interstitial collagen in the underlying stromal tissue, as shown in tissue sections by immunocytochemistry. Using an antibody directed against the purified enzyme, we also showed by immunocytochemistry that the gelatinase was present in tracheal tissue and was specifically located at the periphery of some tracheal gland acini. Northern blots showed higher concentrations of gelatinase A mRNA in glands than in epithelium microdissected from adult cow tracheas. These data indicate that gelatinase A is a specialized product of the tracheal gland epithelial cell, a cell type normally invasive as part of its developmental program; the enzyme may play an important role in normal gland development and disease-associated hypertrophy.

Animals↗

PubMed1982

Structural and functional correlations in the hypertrophic mucosa of intestinal remnants following resection in rats.

Microdissection techniques were applied to determine the absolute increase in villus size and absorptive surface in the mucosa of rat intestinal remnants 3 weeks after 60% proximal resection. Adjacent tissues were used to evaluate the operational kinetic parameters describing the influx of L-phenylalanine and alpha-methyl-D-glucoside into intestinal rings. The villi of ileal remnants were enlarged significantly in all dimensions; the mean surface area of the villus was 2.5 times, and mucosal:serosal surface ratio 1.9 times larger than normal. The influx of phenylalanine and alpha-methyl-glucoside into the mucosa of ileal remnants was reduced, but the change could not be ascribed to an attenuation of one specific kinetic parameter. Using the microdissection data to transform the Vmax-values to a mucosal area basis, we could demonstrate that the Vmax for both substrates was smaller than normal. The results may derive from changes in the maturity of the cell population in intestinal remnants.

Animals↗

PubMed2004

Use of whole genome amplification and comparative genomic hybridisation to detect chromosomal copy number alterations in cell line material and tumour tissue.

We have established that whole genome amplification (WGA), in conjunction with genomic DNA array comparative genomic hybridisation (gaCGH) allows for the identification of genome-wide copy number abnormalities (CNAs) in DNA extracted from both cell line and patient material. To determine the fidelity and reproducibility of WGA to detect copy number imbalances using gaCGH, well characterized cell line genomic DNA was analysed. The gaCGH data obtained from non-amplified DNA and amplified DNA for the neuroblastoma cell line NUB7 and a paediatric medulloblastoma patient was almost identical. In addition, laser capture microdissection (LCM) of prostate tumour cells and subsequent WGA allowed for the detection of a number of CNAs that may not have been identified if DNA had been extracted in bulk from heterogeneous tissue. The results presented here demonstrate the use of WGA for generating sufficient DNA for gaCGH analysis without the introduction of significant sequence representation bias. The combination of amplification and gaCGH using DNA extracted from archival patient material has the potential for permitting the studying of DNA from small cancerous or pre-cancerous foci, which may help to identify potential genomic markers for early diagnosis.

Cell Line↗

PubMed2006

Heterogeneous microsatellite instability observed within epithelium of ulcerative colitis.

Microsatellite instability (MSI) has been associated with colitic cancer. However, reported frequency of MSI was varied and the association of MSI with mismatch repair (MMR) deficiency was unclear. In addition, the occurrence of genetic alterations in stromal cells within ulcerative colitis (UC) is still controversial. We therefore sampled 164 microareas in various pathological lesions of UC with or without colitic cancer and studied the MSI status in relation to the DNA repair protein expressions. A total of 129 microfoci from colorectal tissue of 5 colitic cancer patients and 35 microfoci of 7 UC patients (without neoplasm) were carefully sampled by laser-capture microdissection. MSI was analyzed in each microsamples. The protein expression of MMR genes (MLH1, MSH2, MSH6), O(6)-methylguanine-DNA methyltransferase and p53 were assessed by immunohistochemical analysis. Variety of di-nulcleotide microsatellite markers was altered in individual microfoci from different morphological epithelial lesions, in full range of nonneoplastic epithelium to colitic cancer. Interestingly, MSI was not observed in stromal cells at any sites, including those within colitic cancer/dysplasia lesions. Expression of the MMR proteins was not lost in any of the lesions examined. Microsatellite alterations rather seem to be related to the initiation than to the progression of colitic cancer.

Adolescent↗