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At least 1,081 records · Page 60Linked to original sources
PubMed2001

Alterations of gene expression during colorectal carcinogenesis revealed by cDNA microarrays after laser-capture microdissection of tumor tissues and normal epithelia.

To identify a set of genes involved in the development of colorectal carcinogenesis, we compared expression profiles of colorectal cancer cells from eight tumors with corresponding noncancerous colonic epithelia using a DNA microarray consisting of 9216 human genes. These cell populations had been rendered homogeneous by laser-capture microdissection. Expression change in more than half of the tumors was observed for 235 genes, i.e., 44 up-regulated and 191 down-regulated genes. The differentially expressed genes include those associated with signal transduction, metabolizing enzymes, production of reactive oxygen species, cell cycle, transcription, mitosis, and apoptosis. Subsequent examination of 10 genes (five up-regulated and five down-regulated) by semiquantitative reverse transcription-PCR using the eight tumors together with an additional 12 samples substantiated the reliability of our analysis. The extensive list of genes identified in these experiments provides a large body of potentially valuable information of colorectal carcinogenesis and represents a source of novel targets for cancer therapy.

Colorectal Neoplasms↗
PubMed2001

[Cloning, sequencing and chromosome location of Sry gene of Muntjak munticus vaginalis by DOP-PCR and microdissection].

We isolated 1, Y1, Y2 chromosomes of the male M.m vaginalis by microdissection and amplified the DNA copies by DOP-PCR. Using the DOP-PCR products of 1, Y1, Y2 chromosomes of M.m vaginalis as template respectively and the primers designed within human SRY HMG box, the Sry gene of the male M.m vaginalis was amplified, and it was cloned and sequenced. It is proved that Y2 is the real sex chromosome in the male M.m vaginalis at molecular level for the first time, and Sry was localized on Y2 chromosome.

Animals↗
PubMed2001

Whole genome amplification and high-throughput allelotyping identified five distinct deletion regions on chromosomes 5 and 6 in microdissected early-stage ovarian tumors.

Investigation of genetic changes in tumors by loss of heterozygosity is a powerful technique for identifying chromosomal regions that may contain tumor suppressor genes. In this study, we determined allelic loss on chromosomes 5 and 6 in 29 primary early-stage epithelial ovarian carcinomas including 3 microscopically identified adenocarcinomas using a high-throughput PCR-based method combined with laser capture microdissection and whole genome amplification techniques. Twenty microsatellite markers spanning chromosomes 5 and 6 at an average distance of 20 cM were examined. High frequencies of loss on chromosome 5 were identified at loci D5S428 (48%), D5S424 (32%), and D5S630 (32%). Our study also showed that chromosome 6 exhibited high frequencies of loss of heterozygosity at loci D6S1574 (46%), D6S287 (42%), D6S441 (45%), D6S264 (60%), and D6S281 (35%). These results suggest that multiple tumor suppressor genes are located on five distinct regions on chromosomes 5 and 6, i.e., 5p15.2, 5q13-21, 6p24-25, 6q21-23, and 6q25.1-27, and may be involved in the early development of ovarian carcinomas.

Adenocarcinoma↗
PubMed2001

K-ras mutation at codon 12 in stage I pancreatic adenocarcinoma: analysis by laser capture microdissection and direct sequencing.

Pancreatic ductal adenocarcinoma has been reported to carry a rate mutation high in codon 12 of the K-ras oncogene. To avoid the pitfalls of conventional methods of tissue dissection that might affect the sensitivity and specificity of detecting K-ras mutation, laser capture microdissection (LCM) technique was used. Pancreatic adenocarcinoma tissues were obtained from 15 patients who underwent Whipple's procedure. Selected tissues procured by LCM were analyzed by direct sequencing after polymerase chain reaction amplification of K-ras sequences at codon 12. K-ras mutation was noted in nine patients. All mutations showed G to A substitution at codon 12. The mutational pattern (GGT to GAT) is similar in both western and eastern reports. LCM is a feasible method to effectively obtain pure tumor cells from a surgical specimen. It remains to be determined whether this low mutation rate is a result of relatively early stage of disease or different carcinogenesis in different geographic regions.

Adenocarcinoma↗
PubMedDate not supplied

[A microdissection study of Lampetra fluviatilis lamprey nephrons].

Each of the nephrons in the lamprey L. fluviatilis consists of three distinct segments-proximal, thin, and distal ones. Proximal segments are differentiated into a convoluted and a descending parts, whereas distal ones-into a convoluted and an ascending parts. Therefore microdissection studies indicate that the anatomical composition of a single nephron in the river lamprey is identical to that of a superficial nephron in mammals. Parallel arrangement of the proximal descending, thin, and distal ascending segments, as well as of the collecting tubules, also makes the kidney of the lamprey similar to the countercurrent system in the medulla of mammalian kidney. The data obtained imply that Henle's loop is present in the kidneys not only of higher Vertebrates, but of Cyclostomes as well.

Animals↗
PubMedDate not supplied

Presence of genetic alterations in microdissected stroma of human colon and breast cancers.

BACKGROUND: Human carcinomas not only consist of neoplastic epithelial cells but also of tumor stroma, which may play an important role in tumor-progression. Whereas the tumor surrounding stroma is generally believed to represent a reactive component induced by tumor cell derived factors, a contribution of neoplastic cells to stroma formation via epithelium-mesenchyme transition during tumor invasion has become a novel concept in recent years. MATERIALS, METHODS AND RESULTS: We here show, by laser-assisted microdissection, that frequent genetic alterations in non-hereditary invasive human colon and breast cancers (loss of heterozygosity and TP53 mutations) occur not only in the neoplastic epithelial cells, but also in the adjacent fibroblastic stroma and that both components can share clonal features. CONCLUSION: Tumor cell-mesenchyme transitions are among the possible explanations for these findings and could actually occur during tumor invasion in vivo.

Breast Neoplasms↗
PubMed2001

Microdissection and chromosome painting of plant B chromosomes.

Plant chromosome microdissection techniques together with different isolation and amplification methods of microisolated DNA are described. Such isolated DNA was used to 'chromosome paint' B chromosomes of the dicot Brachycome dichromosomatica and the monocot Secale cereale. It is demonstrated that the specific painting of the described chromosomes was possible because of enrichment for chromosome-specific repetitive sequences, rather than the chromosome specific low- and single-copy sequences which are responsible for the painting of mammalian chromosomes. The feasibility of 'chromosome painting' of standard chromosomes in plant species with relatively small or large genomes is discussed.

Asteraceae↗
PubMed2002

Telomerase activity in microdissected human breast cancer tissues: association with p53, p21 and outcome.

To evaluate the invasive potential of early tumorous lesions of the breast, especially DCIS, we have analyzed semiquantitative telomerase activity in well defined microdissected tissue areas from malignant or benign breast lesions from 145 patients. In order to prove the relationship to cell cycle defects, p53 and p21 proteins were analyzed in corresponding cryostat sections by immunohistochemistry. Telomerase activity was detected in 3 (33.3%) out of 9 benign breast lesions and 109 (80.1%) of 136 malignant tumors. Our study failed to demonstrate an association between telomerase activity, p21, established prognostic factors, such as lymph node status and metastasis, and clinical outcome. We found a high rate of cases (42.2%) with intratumoral heterogeneity concerning telomerase activity status. Telomerase heterogeneity was more obvious in samples with mixed tissue types, although we could not assign specifically the telomerase activity to lobular, ductal and intraductal tissue areas. Telomerase activity was detected in 81.8% of ductal carcinoma in situ (DCIS) which indicates telomerase reactivation as an early event in carcinogenesis. Fifteen (19.5%) out of 77 cases were positive for p53 protein in immunohistochemistry and found to be significantly more frequent in the subgroup with poor outcome. There was only a trend to an association of p53 with high level of telomerase. The prognostic relevance of telomerase activity for patients with benign breast lesions must be further investigated.

Adult↗
PubMedDate not supplied

Gene expression profiles in human BPH: utilization of laser-capture microdissection and quantitative real-time PCR.

Human benign prostatic hyperplasia (BPH) nodules consist of epithelium and stroma that have different properties functionally as well as morphologically. To investigate the molecular profiles of the prostate gland, separate examination of these components are necessary. Laser-capture microdissection (LCM) is a newly-developed device which enables one to dissect interesting parts of tissues under the microscope. In the current work we studied the gene expression profiles of prostatic epithelium and stroma using LCM. Androgen receptor (AR), estrogen receptor alpha and beta (ER alpha and ER beta), progesterone receptor (PR) and 5-alpha reductase type I and type II (5alphaR I and 5alphaR II) gene expressions were studied by RT-PCR. All of these genes were expressed both in the epithelium and stroma. Furthermore, AR transcript was quantified by quantitative real-time PCR. AR transcript ranged from 302 to 1440 copies per 10(5) GAPDH copies and from 257 to 3223 copies per 10(5) GAPDH copies in the epithelium and stroma, respectively. Thus, LCM and quantitative real-time PCR are powerful tools for molecular analysis of heterogeneous tissues including prostate gland.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2002

[Analysis of telomerase activity in esophageal carcinoma using microdissection telomeric repeat amplification protocol assay].

OBJECTIVE: To investigate the changes of telomerase activities in atypical hyperplasia of esophageal mucosal epithelium and esophageal squamous cell carcinoma (SCC) and to study the association of telomerase activity with differentiation, invasiveness, and lymphatic metastasis of cancer. METHODS: The telomerase activities of esophageal SCC tissues, adjacent tissue with dysplasia, and normal mucosal epithelium from surgical edge of 45 cases were detected by microdissection-TRAP (Telomeric Repeat Amplification Protocol)-silver assay. RESULTS: The telomerase activity rates were 79.3% (23/29) in atypical displastic epithelium, 82.2% (37/45) in SCC tissue, and only 5% (2/40) in normal epithelium. The difference of telpmerase activity between dysplastic and normal esophageal epithelia was highly significant (P < 0.01). In the same cancer tissue the differences of telomerase activity among cancer nests to different degrees of defferentiation and to different depths were not significant (P > 0.05). The positive rate of telomerase activity was significantly higher in cases with lymphatic metastasis than in cases without lymphatic metastasis (P < 0.05). CONCLUSION: The telomerase activities is increased in esophageal SCC tissue and adjacent atypical dysplastic tissue. The telomerase activity in SCC tissue is related to lymphatic metastasis but not related to cancer differentiation.

Adult↗
PubMed2002

Compartment analysis of T cell receptor gamma--and immunoglobulin V(H) rearrangements by microdissection in patients with malt lymphoma and chronic gastritis with lymphatic hyperplasia.

The interpretation of clonality within H. pylori-associated gastritis and low-grade MALT lymphoma remains controversial. Due to the observation of MALT lymphoma regression after H. pylori eradication, new definitions concerning the border between benign reactive lesions and malignant gastric lymphoma are needed. Gene rearrangements for immunoglobulin heavy-chain in low-grade MALT lymphoma (N= 12) and H. pylori-associated chronic gastritis with lymphatic hyperplasia (N= 13) were analyzed by microdissection and polymerase chain reaction. Furthermore, T cell receptor-gamma chain rearrangements were analyzed by gene scan analysis. In 11 of 12 cases with initial low-grade MALT lymphoma, intraepithelial and subepithelial B cell rearrangements showed a restricted usage of the immunoglobulin heavy-chain 3. In H. pylori-associated chronic gastritis, the intraepithelial B cell compartment showed an oligoclonal the immunoglobulin heavy-chain rearrangement pattern with a predominance of VH3. The subepithelial compartment did not show any restrictive immunoglobulin heavy-chain gene usage. Additionally a mono- to oligoclonal rearrangement pattern of the T cell receptor-y chain was observed in low-grade MALT lymphoma, whereas an oligoclonal pattem was observed in chronic gastritis. Our data provide evidence that low-grade MALT lymphoma may start within the epithelium and subsequently infiltrate the subepithelial compartment. The observation of a mono- to oligoclonal TCR-gamma rearrangement suggests that an antigen selecting process also takes place within reactive T cells. Combining TCR-gamma gene scan analysis with IgH chain rearrangement analysis might help in discriminating between chronic gastritis and initial MALT lymphoma in questionable cases.

B-Lymphocytes↗
PubMed2002

Laser capture microdissection and microarray expression analysis of lung adenocarcinoma reveals tobacco smoking- and prognosis-related molecular profiles.

Recent expression profile analyses revealed that lung adenocarcinomas can be divided into several subgroups with diverse pathological features. Because cellular heterogeneity of tumors can confound these analyses, we used laser capture microdissection and microarray expression analysis to characterize the molecular profiles of lung adenocarcinomas. We found 45 genes delineating smokers and nonsmokers that were located at chromosomal loci frequently altered in non-small cell lung cancers, and 27 genes, which were differentially expressed between survivors and nonsurvivors 5 years after surgery. These results are consistent with the hypothesis that the abnormal expression of genes involved in maintaining the mitotic spindle checkpoint and genomic stability, e.g., hBUB3, hZW10, and APC2, contribute to the molecular pathogenesis and tumor progression of tobacco smoke-induced adenocarcinoma of the lung.

Adenocarcinoma↗
PubMed2002

Telomerase activity analysis of esophageal carcinoma using microdissection-TRAP assay.

OBJECTIVES: To investigate telomerase activity in esophageal squamous cell carcinoma (SCC) and its preneoplasia lesions, and to study the relationships between telomerase activity and cancer differentiation, cancer invasiveness, and lymphatic metastasis. METHODS: Telomerase activity in esophageal SCC tissues, adjacent dysplasia tissues and normal epithelia from the surgical edge were assessed by microdissection-TRAP (telomeric repeat amplification protocol)-silver staining assay. RESULTS: Telomerase activity was detected in 37 (82.2%) of 45 esophageal tumors, 23 (79.3%) of 29 dysplasias, and 2 (5%) of 40 normal epithelia. There was a significant difference in activity between dysplasia and normal epithelium, as well as between tumor and normal epithelium. Twenty-six (92.9%) of 28 tumors with lymphatic metastasis had detectable telomerase activity compared to 11 (64.7%) of 17 non-lymphatic metastasis tumors. These relationships were statistically significant (P < 0.05), but the one between telomerase activity and tumor grade was not. CONCLUSION: Telomerase activity was high both in esophageal SCC and their preneoplasia lesions. The telomerase activity in SCC tissue was related to lymphatic metastasis, but not to cancer differentiation.

Adult↗
PubMedDate not supplied

In situ gene expression analysis of cancer using laser capture microdissection, microarrays and real time quantitative PCR.

The simultaneous development of laser capture microdissection (LCM) and high-throughput mRNA analysis platforms has provided a significant technological advance in the world of cancer biology. The combination of such technologies provides a unique and powerful opportunity to directly assess the in situ molecular genetic events that are associated with initiation and progression of human malignancies. Despite these technological advances, the integration of LCM with high-throughput gene expression analysis has been met with various challenges. The goal of this review is to highlight some of the obstacles that we have faced and continue to face as we try to optimally apply LCM to in situ gene expression analysis.

Gene Expression Regulation, Neoplastic↗
PubMed2002

Extensive somatic mitochondrial mutations in primary prostate cancer using laser capture microdissection.

Prostate cancer is the second leading cause of cancer deaths among men in the United States,but the precise molecular events leading to prostate carcinogenesis are not well understood. We isolated histologically defined cell populations from prostate cancer and its preinvasive lesions using laser capture microdissection, and performed genetic analysis on the mitochondrial genome, a sensitive cytoplasmic DNA. An extremely high incidence of somatic mutation (90% of prostatectomy cancer specimens) was found in the control region (the displacement loop) of mitochondrial DNA. The massive induction of lesion-associated mutations suggests active mitochondrial mutagenesis in both prostate cancer and its preinvasive lesions. Inspection of these mutations provides new insights into prostate cancer genetics and reveals unique patterns of somatic mutations in prostatic neoplastic lesions.

Aged↗
PubMed2002

[Application of laser capture microdissection in screening phage-display peptide library from osteosarcoma tissues].

OBJECTIVE: To establish a novel approach by using laser capture microdissection (LCM) for phage-display peptide library screening and direct recovery of the peptides specifically bound to the cells from fresh human tissue. METHODS: Fresh human osteosarcoma (OS) tissue specimens obtained from biopsy were incubated in phage peptide library solution, followed by shaking at 4 degrees celsius and subsequent preparation of frozen sections. Immunohistochemistry was employed to examine the infiltration of the phage in the tissues. After modification to improve the viability of the phage during the operation, LCM was used to isolate the targeted tumor cells that specifically bound the phage peptides. The phage was then recovered by transfecting the host bacteria, and the specificity of the selected phage was assessed by calculating the recovered phage transfection unit on different tissue sections. RESULTS: Enough phages that specifically bound to tumor cells were recovered from the sections. After three rounds of screening, the eluted phage showed up to 16-fold selectivity for OS tissue. CONCLUSION: Application of LCM makes it possible to screen phage display peptide library directly from fresh human tissue. Specific cell-binding peptides can be selected directly from fresh human tumor cells in their native tissue environment at the single cell level.

Animals↗
PubMedDate not supplied

Gene expression profiles of human BPH (II): Optimization of laser-capture microdissection and utilization of cDNA microarray.

Laser capture microdissection (LCM) enables the dissection of heterogeneous components of tissues, helping to investigate the molecular properties of these tissues. We have reported gene expression profiles in human benign prostatic hyperplasia (BPH) using LCM and quantitative real-time PCR. In the current work, we extended the previous observations. Firstly, we studied the relationship between the number of dissected acini and amplification by PCR, and found that androgen receptor (AR) and 18S rRNA transcripts were successfully amplified from total RNA obtained from one prostate acinus. Furthermore, LCM-dissected samples were applicable to methylation-specific PCR of E-cadherin promoter gene after bisulfite modification of genomic DNA. Next, we performed cDNA microarray analysis to screen gene expression profiles in the epithelium and stroma. RNA was amplified by T7-RNA polymerase and labeled with Cy3 and Cy5. Epithelium-related or stroma-related genes were identified through cDNA microarray. We confirmed true gene expression levels by quantitative real-time PCR. In epithelium, E-cadherin and serine protease 2, Kunitz-type gene expression levels were significantly elevated, while the connective tissue growth factor gene expression level was significantly elevated in stroma. Thus, LCM-dissected samples were applicable to various molecular examinations including methylation-specific PCR and cDNA microarray, and this will contribute to a precise understanding of the molecular profiles of prostate glands.

DNA↗
PubMed2001

[Loss of heterozygosity on chromosome 9p13-23 in microdissected laryngeal squamous cell carcinoma by microsatellite analysis].

OBJECTIVE: Loss of heterozygosity (LOH) studies indicate that allelic loss associated with the development of head and neck of squamous cell carcinoma occurs most frequently at 9p21-22. However, the target of chromosome 9p21-22 loss has been the source of significant debate. A putative tumor suppressor gene, p16, has been identified at the 9p21 location, but genetic alterations of p16 located in this region are unusual. To refine the hot spots of LOH on chromosome 9p13-23 will be helpful to find other putative tumor suppressor genes in laryngeal squamous cell carcinoma. METHODS: The sections of paraffin-embedded tumor tissues were microdissected to enrich for neoplastic cells. LOH on 9p13-23 was analyzed in a set of 42 paired blood and tumor samples using polymerase chain reaction (PCR) with 13 highly polymorphic microsatellite markers. RESULTS: Of the 42 total tumors, 41(97.6%) showed LOH in at least one of the microsatellite markers tested at the chromosome 9p13-23 region. The most frequently deleted marker was D9S162 with in 17 of 19 (89.5%) informative samples. The marker D9S171, which is located in 9p21, had LOH detected in 12 of 15 informative cases (80.0%). LOH at the D9S1748 marker (closest to the p16 gene locus) was detected in 18 of 36 informative cases (50.0%). Fine deletion mapping also revealed two minimal regions of LOH encompassing markers D9S161-D9S171 at 9p21 and IFNA-D9S162 at 9p22-23. CONCLUSION: These findings imply the high frequency of LOH at 9p13-23 in laryngeal squamous cell carcinoma and the presence of at least two as yet unidentified tumor suppressor genes in 9p13-23 region. Those putative tumor suppressor genes may become inactivated during the progression of the laryngeal squamous cell carcinoma.

Adult↗