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PubMed2005

Selective heparanase localization in malignant melanoma.

Heparanase (HPSE-1) is an endo-beta-D-glucuronidase that cleaves heparan sulfate proteoglycans (HSPG), and its expression has been associated with increased growth, metastasis, and angiogenesis of tumors. Since metastatic melanoma cells express high levels of HSPG and because melanoma tumors grow highly vascularized, we analyzed melanoma tissue specimens for HPSE-1 expression from experimental animals as well as from patients. Laser capture microdissection microscopy was used to extract melanoma cell populations and to isolate them from adjacent tissue. In experimental animals, a 29-fold upregulation of HPSE-1 expression was detected by real-time PCR in metastatic melanoma compared to normal lung tissue. Additionally, immunohistochemistry (IHC) revealed selective HPSE-1 staining in human metastatic melanoma when compared to primary melanoma tumors from the same patient. IHC also showed a marked staining for the enzyme around blood vessels and in vascularized regions. Our results provide evidence demonstrating that HPSE-1 likely plays important roles in regulating the in vivo growth and progression of melanoma. These results further emphasize the importance that therapies designed to block HPSE-1 activity may aid in controlling this type of cancer.

Animals↗
PubMed2001

Gene expression in a pure population of odontoblasts isolated by laser-capture microdissection.

Studies of odontoblast differentiation and function have been limited due to difficulties in obtaining sufficient numbers of intact cells. We describe a novel approach of laser-capture microdissection to obtain homogenous populations of pre-odontoblasts and odontoblasts from tissue sections of mouse molar cusp tips. Fixation, processing, and staining conditions were assessed for the optimal retrieval of total RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA from three-day-old captured odontoblasts was sufficient in quantity and quality. Odontoblast-specific gene expression was studied by RT-PCR analysis performed in a single streptavidin-coated tube. At E15.5, Days 0 and 3, gene expression in laser-captured odontoblasts resembled that seen in vivo by in situ hybridization. The use of LCM is thus a valuable means of retrieving quality RNA from discrete populations of odontoblasts at different stages of dentinogenesis.

Animals↗
PubMed2005

Aberrant hypermethylation of RASSF1A promoter in ovarian borderline tumors and carcinomas.

The newly identified 3p21.3 tumor suppressor gene RASSF1A is inactivated by hypermethylation in variable solid tumors, including those of the lung, breast, prostate, kidney, and ovary. The purpose of this study was to evaluate the methylation status of RASSF1A in various types and stages of ovarian epithelial tumors. We analyzed the DNA methylation status of ovarian tumors using methylation-specific polymerase chain reaction in 54 frozen ovarian tumor tissues and in 97 cases of archival ovarian serous epithelial tumors using a microdissection procedure. Hypermethylation statuses were examined vs clinicopathologic findings. RASSF1A promoter methylation rates in the various types of fresh ovarian tissues were as follows: serous cystadenoma (1/5), serous tumor of borderline malignancy (2/7), serous adenocarcinoma (4/10), mucinous cystadenoma (0/5), mucinous tumor of borderline malignancy (2/7), mucinous adenocarcinoma (3/6), transitional-cell carcinoma (1/3), clear-cell carcinoma (3/3), and malignant müllerian mixed tumor (3/3). In archived serous tumor tissues, RASSF1A promoter hypermethylation was detected in serous cystadenoma (1/6, 16.6%), serous tumor of borderline malignancy (20/41, 48.8%), and in serous adenocarcinoma (25/50, 50%). The status of RASSF1A hypermethylation in borderline tumors was found to correlate statistically with the presence of microinvasion (p=0.002), peritoneal implant (p<0.001), and bilaterality (p=0.019). The RASSF1A promoter hypermethylation was frequently found in borderline tumors and carcinomas, suggesting that RASSF1A promoter hypermethylation may be a useful molecular marker for the early detection of ovarian tumors.

Adenocarcinoma↗
PubMed2003

Single-cell immunohistochemical mutation load assay (SCIMLA) using human paraffin-embedded tissues.

It would be advantageous to measure mutation load in situ in order to determine the relationship between a high mutation load and increased risk for cancer or other diseases and to evaluate sources of possible mutagen exposure. Previously, in situ mutation detection assays have been plagued with multiple rounds of amplification and high rates of false-positives and false-negatives. The single cell immunohistochemical mutation load assay (SCIMLA) was developed to measure somatic mutation frequency, pattern, and spectrum in normal tissues with a single round of amplification. The P53 gene was utilized as a mutation reporter because of the unusual property that missense mutations often cause P53 protein to accumulate in the cell, allowing the mutant proteins to be detected by immunohistochemical staining. Alternative reporter genes with stabilized mutant proteins may be envisioned. Single cells that stain positively for P53 protein overabundance (red cells) were microdissected from ethanol-fixed and paraffin-embedded tissues. A novel stimulated-PCR (S-PCR) protocol permitted successful amplification of a 1.8-kb segment of the P53 gene (i.e., exons 5-9) in 87% of single mammary cells. Subsequent sequence analysis demonstrated that 35% of the amplified red-stained epithelial cells from normal breast tissue have missense mutations at evolutionarily conserved amino acids. Jackpot mutations, presumably due to clonal expansion, were common. False-positive missense mutations at conserved residues were observed in 3% of the clear cells (i.e., without red stain), presumably due to DNA polymerase error in early PCR cycles. The allele dropout rate was measured at 40% of the amplified cells. SCIMLA is applicable to a variety of tissues, utilizes a single amplification of an endogenous gene, displays mutant cells in situ, and may be adapted to other species.

Base Sequence↗
PubMed2003

Resolution of clonal origins for endometriotic lesions using laser capture microdissection and the human androgen receptor (HUMARA) assay.

OBJECTIVE: To determine the clonal origins of endometriotic lesions using laser capture microdissection and PCR-based HUMARA assay. DESIGN: Molecular genetic study of human tissue. SETTING: Molecular genetics laboratory in an academic setting. PATIENT(S): Twenty patients with endometriosis. Forty specimens of endometriotic lesions from these patients and one specimen of normal endometrium were analyzed. INTERVENTION(S): Laser capture microdissection was used to harvest epithelial cells from single and multifocal endometrial lesions from paraffin-embedded and frozen tissues, and their clonality was determined with the HUMARA assay. MAIN OUTCOME MEASURE(S): Polymerase chain reaction-based HUMARA assay of clonality. RESULT(S): Thirty-eight specimens were polymorphic and thus informative. Most specimens were monoclonal, as determined by the HUMARA assay. In four specimens of multifocal lesions, polyclonality was detected, but upon more refined microdissections and further analyses, we found that each focus was monoclonal individually. CONCLUSION(S): Previously reported polyclonality is very likely to be attributed to the pooling of multifocal lesions or contamination of normal tissues. These results suggest that endometriotic lesions were monoclonal in origin, and in the case of multifocal lesions, each focus originates monoclonally; hence, different foci have independent origins. The monoclonality of endometriotic lesions suggests that they may carry neoplastic potentials, and the apparent independent origins of multifocal lesions suggest that reconstruction of individual lesion histories may help us to understand the initiation and progression of endometriosis.

Adult↗
PubMed2004

[Detection of IgH gene rearrangement in Reed-Sternberg cells microdissected from classical Hodgkin's lymphoma].

OBJECTIVE: To study the origins and clonality of Hodgkin and Reed-Sternberg (H/RS) cells and their relations with the background lymphocytes in classical Hodgkin's lymphoma. METHOD: IgH gene rearrangement was detected in paffin-embedded tissues from 33 patients with Hodgkin's lymphoma and a further analysis of the gene rearrangement was conducted in 6 of the positive cases identified after immunostaining of the sections with B-cell-specific activator protein (BSAP) followed by microdissection of the positivity labeled H/RS cells and background lymphocytes. RESULTS: IgH gene rearrangement was identified in 16 of the 33 cases. Microdissection of the lymphoma tissues was successfully performed in the 6 positive cases, and of the 19 tubes of H/RS cells obtained, 14 presented clonal bands of the rearrangement, and difference in cell numbers did not significantly influence the positive rate (P=0.290); in the 12 tubes of microdissected background lymphocytes obtained, 2 were positive for the rearrangement, and the positive rates for the rearrangement significantly differed between H/RS cells and the background lymphocytes (P=0.002). CONCLUSION: The results appear to support the hypothesis that H/RS cells originates from B cells, and a part of the background lymphocytes may possess neoplastic proliferation potentials to function as the precursors of H/RS cells.

Adolescent↗
PubMed1997

DNA extraction from paraffin-embedded tissues using a salting-out procedure: a reliable method for PCR amplification of archival material.

Many techniques have been described for the extraction of DNA from paraffin-embedded tissues. Numerous efforts have been directed at simplification of these methods for rapid analysis using PCR. One disadvantage to some of the simpler procedures is inefficient PCR amplification, and for more involved ones using phenol/chloroform extraction, reduction in the yield of DNA. In the present study we report the use of a novel salting-out procedure that was utilized to extract DNA from 259 separate microdissection specimens of formalin-fixed, paraffin-embedded tissue sections. These sections were derived from 97 patients with tumors of the ampulla of Vater resected between 1965 and 1995 at our institution. The mean DNA yield was 22.75 micrograms (median 13.2 +/- 30.25) and the mean 260/280 absorbance ratio was 1.68 (median 1.70 +/- 0.25). All specimens (259/259) were successfully used to amplify K-ras exon 1 by a nested PCR technique. These results indicate that this DNA extraction method produces good yields of quality DNA, even from specimens several decades old.

Ampulla of Vater↗
PubMed2005

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.

Carcinoma, Hepatocellular↗
PubMed2006

Immobilization stress induces BDNF in rat submandibular glands.

Brain-derived neurotrophic factor (BDNF) promotes survival and differentiation of the cells of the central and peripheral nervous systems. BDNF has been identified in non-neural tissue, including the heart, lung, platelets, lymphocytes, and lacrimal glands. Immobilization stress modifies BDNF mRNA expression in some organs. The present study examines the effect of immobilization stress on BDNF, and its receptor TrkB, in male rat submandibular glands. Increased BDNF mRNA and protein expression were observed in duct cells as a result of immobilization stress, as demonstrated by real-time PCR, Western blot, immunohistochemistry, and analysis by microdissection. TrkB mRNA was not detected in salivary gland tissue, or oral or esophageal mucosa, by RT-PCR. Rat submandibular gland was thus identified as an organ which expresses BDNF. Furthermore, the results of this study suggest that increased salivary BDNF expression occurs following immobilization stress.

Animals↗
PubMed1996

Hepatitis C virus sequences encoding truncated core proteins detected in a hepatocellular carcinoma.

RNA was specifically extracted from tumor and peritumor tissue of a hepatitis C virus (HCV)-associated hepatocellular carcinoma after microdissection. RT-PCR products of the HCV 5'-noncoding (NC), core and nonstructural (NS)-5 gene were examined. Nucleotide sequences of the core region derived from the tumor tissue revealed deletions and mutations resulting in truncated proteins. Peritumor and serum HCV sequences were unaffected.

Amino Acid Sequence↗
PubMed1997

Molecular genetic evidence for the conversion hypothesis of the origin of malignant mixed müllerian tumours.

The origin of malignant mixed Müllerian tumours (MMMTs) has long been debated, due to the indefinite relationship between epithelial and mesenchymal malignant cells. In order to obtain insight into the clonal relationship between the two components of these tumours, molecular genetic changes were investigated at the level of loss of heterozygosity (LOH) in both cells types. LOH was studied in a series of six cases with 74 polymorphic microsatellite markers mapping to 19 different chromosomes. The epithelial and the mesenchymal neoplastic cells were separately microdissected from formalin-fixed, paraffin-embedded tissue, prior to DNA isolation. LOH was observed for 35 different markers mapping to chromosomes 3, 6, 8, 11, 15, 16, 17, 18, 21, and X. The most frequently involved chromosomes were 17p, 17q, 11q, 15q, and 21q. LOH was observed in five out of six cases and identical alleles were lost in the epithelial and in the mesenchymal cells. No genetic differences were observed between the two cell types for any of the informative markers. Immunohistochemistry (IHC) and TP53 mutation analysis revealed involvement of TP53 in all cases. Mutations were identified in five MMMTs. In four tumours, of which three had a missense mutation, strong nuclear staining for p53 was observed. In the remaining two cases, the mutation resulted in a stop codon, with no nuclear staining for p53 by IHC. The results support a monoclonal origin of MMMTs, with the absence of genetic changes uniquely associated with either of the phenotypes. The latter finding is compatible with current opinion that these neoplasms should be considered as metaplastic carcinomas and supports the conversion hypothesis.

Aged↗
PubMed1999

Identification of distinct regions of allelic loss on chromosome 13q in nasopharyngeal cancer from paraffin embedded tissues.

Our main purpose was to identify tumor suppressor gene loci on chromosome 13 responsible for nasopharyngeal cancer (NPC) development by analyzing loss of heterozygosity (LOH) and RB protein expression in paraffin embedded tissues. Normal and tumor DNA were extracted from microdissected samples, and their whole genomes were amplified using degenerate oligonucleotide primers. The polymerase chain reaction (PCR) products were analyzed by repeated amplification using primers derived from 16 microsatellite regions spanning the long arm of this chromosome. Among 50 informative cases, LOH was observed in 44 tumors. Thirty-one tumors displayed partial loss and provided an informative basis for detailed deletion mapping. Three minimal regions of loss were delineated; the first flanked by D13S120 and D13S219, the second by D13S126 and D13S119, and the third by D13S137 and 13qter. These 3 regions were linked to BRCA2 on 13q12, RB1 on 13q14, and 13q14.3-ter, respectively. Seven and 4 cases showed LOH either on 13q12 or 13q14, respectively. Nineteen cases showed LOH of both loci separately. One NPC displayed 13q12 and 13q14.3-ter LOH. RB protein expression was detectable in 76% of the cases. Ten out of 15 cases with the allelic losses limited to 13q14 showed RB protein expression. Contrasting that, 6 out of 7 cases devoid of RB protein expressions showed 13q14LOH. In conclusion, 13qLOH, involving 3 tumor suppressor gene loci, appears to be a frequent genetic event occurring during NPC development. However, other tumor suppressor genes besides RB1, may be responsible for the majority of 13q14LOH.

Adult↗
PubMed2005

AGR2, an androgen-inducible secretory protein overexpressed in prostate cancer.

AGR2, the human homologue of Xenopus anterior gradient 2 (XAG2), was identified by a suppression subtractive hybridization-based technique as an androgen-inducible gene. There are two AGR2 transcripts, which encode the same secretory protein of 175 amino acids. The androgen induction was time- and dose-dependent, with more than a 10-fold increase in the level of AGR2 mRNA after 48 hr of treatment with 10(-9) M R1881. Expression of AGR2 mRNA was specifically detected in limited human tissue rich in epithelial cells, including the prostate gland. Analysis of 46 microdissected primary prostate adenocarcinoma samples showed that AGR2 mRNA expression was markedly elevated in the majority of tumors as compared to matched adjacent benign tissues. Androgen-induced AGR2 protein expression was demonstrated in LNCaP cells by Western blot analysis with an anti-AGR2 antibody. Immunohistochemistry analysis indicated that AGR2 protein expression was highly restricted to the secretory epithelial cells in the prostate gland. In tissue sections from radical prostatectomy specimens, immunohistochemical staining of AGR2 showed markedly increased expression in high-grade prostatic intraepithelial neoplasia and Gleason pattern 3-4 prostatic adenocarcinoma. Therefore, the androgen-induced secretory protein AGR2 may serve as a potential therapeutic target and/or molecular marker for prostate cancer.

Adenocarcinoma↗
PubMed2002

Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity.

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation-sensitive single-strand conformation analysis (MS-SSCA) and direct sequencing using bisulfite-modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p < 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT-expressing telomerase-positive samples and was absent in telomerase-negative samples (p < 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression.

DNA Methylation↗
PubMed2006

Methylation of APC, TIMP3, and TERT: a new predictive marker to distinguish Barrett's oesophagus patients at risk for malignant transformation.

Barrett's associated oesophageal adenocarcinoma (EAC) is one of the most rapidly increasing malignancies in Western countries. Because of its poor prognosis, management of this disease through screening of Barrett's oesophagus (BE) patients and identification of those with a high risk of developing an adenocarcinoma seems a promising approach. Early molecular markers of malignant transformation might contribute to such screening approaches. Gene promoter methylation analysis was performed on normal, pre-neoplastic, and neoplastic lesions from BE patients. All lesions of interest were sampled by microdissection from formalin-fixed paraffin-embedded tissue sections. We found that, in 27 adenocarcinomas, APC, TIMP3, TERT, CDKN2A, and SFRP1 promoters were methylated in 93%, 65%, 64%, 48%, and 91%, respectively; in contrast MLH1, RASSF1, RARB, CDH1, and FHIT promoters were methylated in less than 5% of the tumours. In BE mucosa from patients who had progressed to adenocarcinoma (12 samples), APC, TIMP3, and TERT promoters were hypermethylated in 100%, 91%, and 92% of cases, whereas in BE mucosa from patients who had not progressed (16 samples) methylation was found only in 36%, 23%, and 17%, respectively. Furthermore, the epigenetic profile of BE with and without EAC differed significantly with, respectively, 81% and 26% of the PCR samples showing promoter hypermethylation for APC, TIMP3, and TERT (p < 0.0001). Promoter methylation of CDKN2A was infrequently detected in BE samples, while SFRP1 methylation was observed in all samples. Our results suggest that promoter methylation profiling of BE using multiple target genes including APC, TIMP3, and TERT might be used as a predictive marker for increased EAC risk.

Adenocarcinoma↗
PubMed1988

Somatostatin in the idiopathic inflammatory bowel diseases.

To study the effect of mucosal inflammation on tissue concentrations of somatostatin, the distribution and concentration of somatostatin in specimens of normal and abnormal (ulcerative colitis and Crohn's disease) ileum and colon were determined by a specific radioimmunoassay. Each tissue specimen obtained at surgery was separated by microdissection into the mucosa-submucosa and the muscularis externa. Immunoreactive somatostatin was acid-extracted from each layer before measurement. Gel chromatography was used to characterize immunoreactive somatostatin measured by radioimmunoassay; somatostatin-28 was the major immunoreactive species measured in human intestine. In normal colon, concentrations of somatostatin were not related to patient age. Concentrations of immunoreactive somatostatin in the mucosa-submucosa of the descending colon were significantly decreased in ulcerative colitis and in Crohn's colitis, compared with normal colon. There was no apparent relationship between concentrations of somatostatin and the duration of inflammatory bowel disease. However, somatostatin concentrations appeared to be lower in patients with severe colitis than in patients with minimal colitis. The decrease in mucosal-submucosal concentrations of somatostatin is in agreement with previous morphologic studies, which have suggested diminished populations of endocrine cells in ulcerative colitis. The possible role of somatostatin in the colon suggests that further studies of the alteration of this gut peptide may be useful in understanding a component of the pathophysiology of idiopathic inflammatory bowel disease.

Chromatography, Gel↗
PubMed2002

Detection of gene expression of vascular endothelial growth factor and flk-1 in the renal glomeruli of the normal rat kidney using the laser microdissection system.

The recent development of the laser microdissection (LMD) technique enables one to target particular tissues or cells for gene or protein analyses. The purpose of this study was to detect local mRNA expression of vascular endothelial growth factor (VEGF) and its receptor, flk-1, in the glomeruli of normal rat kidneys using the LMD system. Frozen sections of the kidney of 8-week-old male Wistar rats were made. The glomeruli were dissected from the frozen sections with the LMD system, and total RNA was extracted from 200 glomeruli in each kidney. Reverse-transcription polymerase chain reaction (RT-PCR) revealed the local mRNA expression of three isoforms of VEGF, flk-1 and GAPDH in the glomeruli. Moreover, the real-time PCR was performed to evaluate the experimental condition for quantification of VEGF and flk-1 mRNA expression using this system, and the results showed that at least 10 glomeruli might be needed for quantifying local VEGF mRNA expression. However, cDNA from 200 glomeruli was not enough for quantitative evaluation of flk-1 mRNA with this system. These results demonstrate the reproducibility of the analysis of mRNA expression in the renal glomeruli using the LMD system and also suggest that the application of the LMD technique will provide information to further our understanding of the mechanisms involved in kidney diseases.

Angiogenesis Inducing Agents↗
PubMed1998

Molecular characterization of intraductal breast carcinomas.

In situ duct carcinoma (DCIS) is a heterogeneous group of lesions which has recently been subdivided into three types: well-differentiated (type I), intermediately differentiated (type II) and poorly differentiated (type III) DCIS. Fourteen cases of DCIS and 11 of DCIS with minimal invasion were analysed for mRNA levels of beta-actin, EGFR, c-cerbB2, MTS1, k-ras, RB, BRCA1, cyclin E, and c-myc genes. A microdissection technique was used on paraffin-embedded tissue. A statistically significantly higher expression of cyclin E oncogene and MTS1 tumor suppressor gene was seen in type III DCIS than in the other types, while no significant differences in the mRNA expression patterns of the other genes were observed. These data are consistent with the fact that poorly differentiated DCIS is a readily recognizable class of tumours that have a particularly aggressive behaviour and probably unique histogenesis.

Biomarkers, Tumor↗