Combining Laser Capture Microdissection Research Articles

Unique clockwork in photoreceptor of rat

In mammals, the retina contains a clock system that oscillates independently of the master clock in the suprachiasmatic nucleus and allows the retina to anticipate and to adapt to the sustained daily changes in ambient illumination. Using a combination of laser capture micro-dissection and quantitative PCR in the present study, the clockwork of mammalian photoreceptors has been recorded. The transcript amounts of the core clock genes Clock, Bmal1, Period1 (Per1), Per3, Cryptochrome2, and Casein kinase Iε in photoreceptors of rat retina have been found to undergo daily changes. Clock and Bmal1 peak with Per1 and Per3 around dark onset, whereas Casein kinase Iε and Cryptochrome2 peak at night. As shown for Clock, Per1, and Casein kinase Iε, the oscillation of transcript amounts results in daily changes of the protein products. The in-phase oscillation of Clock/Bmal1 with Pers and the rhythmic expression of Casein kinase Iε do not occur in molecular clocks of other tissues including the suprachiasmatic nucleus. Therefore, the findings presented suggest that the photoreceptor clock is unique not only in its position outside the clock hierarchy mastered by the suprachiasmatic nucleus, but also with regard to the intrinsic rhythmic properties of its molecular components.

MicroRNA‐21 protects neurons from ischemic death

MicroRNAs are small RNAs that attenuate protein expression by complementary binding to the 3'-UTR of a target mRNA. Currently, very little is known about microRNAs after cerebral ischemia. In particular, microRNA-21 (miR-21) is a strong antiapoptotic factor in some biological systems. We investigated the role of miR-21 after stroke in the rat. We employed in situ hybridization and laser capture microdissection in combination with real-time RT-PCR to investigate the expression of miR-21 after stroke. In situ hybridization revealed that miR-21 expression was upregulated in neurons of the ischemic boundary zone, and quantitative real-time RT-PCR analysis revealed that stroke increased mature miR-21 levels by approximately threefold in neurons isolated from the ischemic boundary zone by laser capture microdissection as compared with homologous contralateral neurons 2 days (n = 4; P < 0.05) and 7 days (n = 3; P < 0.05) after stroke. In vitro, overexpression of miR-21 in cultured cortical neurons substantially suppressed oxygen and glucose deprivation-induced apoptotic cell death, whereas attenuation of endogenous miR-21 by antisense inhibition exacerbated cell death after oxygen and glucose deprivation. Moreover, overexpression of miR-21 in neurons significantly reduced FASLG levels, and introduction of an miR-21 mimic into 293-HEK cells substantially reduced luciferase activity in a reporter system containing the 3'-UTR of Faslg. Our data indicate that overexpression of miR-21 protects against ischemic neuronal death, and that downregulation of FASLG, a tumor necrosis factor-α family member and an important cell death-inducing ligand whose gene is targeted by miR-21, probably mediates the neuroprotective effect. These novel findings suggest that miR-21 may be an attractive therapeutic molecule for treatment of stroke.

Cell proliferation and apoptosis in the primary enamel knot measured by flow cytometry of laser microdissected samples

Laser capture microdissection (LCM) uniquely allows the selection of specific cell populations from histological sections. These selected cells are then catapulted into a test tube without any contamination from surrounding tissues. During the last ten years, many significant results have been achieved, particularly at the level of DNA and RNA where amplification techniques are available. However, where amplification procedures are difficult, the benefits of LCM diminish. To overcome such difficulties, a novel approach, combining laser capture microdissection and flow cytometry, has been tested here for detection of apoptosis and proliferation in tissue bound cell populations without any amplification steps. The mouse cap stage molar tooth germ was used as a model. At the centre of the inner enamel epithelium, the primary enamel knot is a clearly defined apoptotic population with minimal proliferation, flanked by the highly proliferative cervical loops on each side. Thus within the tooth germ epithelium at this stage, two distinct populations of cells are found side by side. These populations were selected by laser capture microdissection and then analysed by flow cytometry for apoptosis and proliferation. Flow cytometric results correlated well with immunohistochemical findings, demonstrating the success and sensitivity of this combined procedure.

Repair of Bone Defect Using Bone Marrow Cells and Demineralized Bone Matrix Supplemented with Polymeric Materials

We present a novel, reverse thermo-responsive (RTR) polymeric osteogenic composite comprising demineralized bone matrix (DBM) and unmanipulated bone marrow cells (BMC) for repair of bone defects. The polymers investigated were low viscosity aqueous solutions at ambient temperature, which gel once they heat up and reach body temperature. Our goal to supplement DBM-BMC composite with RTR polymers displaying superior rheological properties, was to improve graft integrity and stability, during tissue regeneration. The osteogenic composite when implanted under kidney capsule of mice, proved to be biocompatible and biodegradable, with no residual polymer detected in the newly formed osteohematopoietic site. Implantation of the osteogenic composite into a large area of missing area of parietal bone of the skull of rats, resulted in an extensive remodeling of DBM particles, fully reconstituted hematopoietic microenvironment and well integrated normal flat bone within thirty days. The quality and shape of the newly created bone were comparable to the original bone and neither local or systemic inflammatory reactions nor fibrosis at the junction of the new and old calvarium could be documented. Furthermore, combined laser capture microdissection (LCM) technique and PCR analysis of male BMC in female rats confirmed the presence of male derived cells captured from the repaired/ regenerated flat bone defect. The use of active self sufficient osteogenic DBM-BMC composite supported by a viscous polymeric scaffold for purposive local hard tissue formation, may have a significant potential in enhancement of bone regeneration and repair following trauma, degenerative or inflamatory lesion, iatrogenic interventions and cosmetic indications.

Quantitative cellular level analysis of mitochondrial DNA 3243A > G mutations in individual tissues from the archival temporal bones of a MELAS patient

Conclusion: We could represent the first quantitative analysis of the mutation rate at the cellular level in human inner ear of a patient with MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) by combining laser capture microdissection (LCM) and quantitative real time PCR. Objectives: We previously reported combining LCM and PCR to isolate mtDNA from the cells of specific tissues within a human archival celloidin-embedded temporal bone section without known otological history. Using this method, we quantitatively analyzed the rate of mtDNA 3243A > G mutation in the inner ear of a MELAS patient, and examined the correlation of the mutation rate at the cellular level and their histopathological condition. Methods: We extracted each inner ear organs using LCM from temporal bone sections of a MELAS patient, and studied the mutation rate, which was calculated as the ratio of the amount of mutant mtDNA to the total mtDNA. Results: We found that the mtDNA mutation rate was high in spiral ganglion cells and the saccular macula, but was comparatively low in hair cells of the organ of Corti, the stria vascularis and the facial nerve. With the exception of the stria vascularis, there was a good correlation between the mutation rate and the histological findings.

Combining laser capture microdissection and proteomics: Methodologies and clinical applications

Laser capture microdissection (LCM) has become an important tool for biomedical research. Various molecular biology techniques have been combined with LCM to reveal molecular profiles at an unprecedented tissue resolution. Proteomics is among those techniques that have proved fruitful in combination with LCM. This review provides an overview of the various proteomic techniques that have been developed for analyzing cells obtained using LCM. Methods for analyzing microdissected cells for various proteomic techniques are described and compared. In addition, various applications based on LCM and proteomics are discussed.

213 ANALYSIS OF STEROID HORMONE RECEPTOR GENE EXPRESSION IN THE BOVINE OVARY AFTER LASER-CAPTURE MICRODISSECTION

Developmental processes in complex organs like the ovary are difficult to study in terms of a biochemical and molecular biological analysis. Laser-assisted microdissection allows the efficient and precise capture of single cells or groups of cells of an organ within the context of time and space and permits their subsequent molecular characterization. Together with real-time PCR techniques, it is now feasible to study gene expression in defined cell populations of complex tissues, but it is essential to create standards optimized for fixation, preparation, and isolation of RNA, reverse transcription reaction, and real-time PCR protocol for every tissue of interest. The aim of our study was to develop protocols for a precise analysis of estrogen receptor alpha (ER-α) and progesterone receptor (PR) in defined compartments of the ovary (granulosa cells, theca interna cells, zona vasculosa, and zona parenchymatosa of the stroma). Additionally, the receptor proteins were localized by immunohistochemistry. A special focus was put on the question of how formalin fixation and paraffin embedding influences the quality of the isolated RNA from microdissected material, which was used for quantitative reverse transcription-PCR (qRT-PCR). Quality and quantity of total RNA extracted from formalin-fixed, paraffin-embedded (FFPE) sections and from material immersed in RNAlater® (Ambion, Foster City, CA, USA) was checked using an Experion automated electrophoresis system (Bio-Rad, Munich, Germany). The RNA quality indicator for microdissected material was between 6 and 7, and for RNAlater® material was 9 or better. Online qRT-PCR using the iCycler SYBR GreenTM protocol (Bio-Rad) was performed in a 96-well plate. Primer pairs were chosen to generate PCR products between 100 bp (ER-α) and 140 bp (PR), as RNA recovered from FFPE-laser microdissected material was expected to be considerably fragmented. Using GenEx software (BioEPS, Freisling, Germany), we showed that the expression of mRNA for PR was much stronger in the theca interna than in the 3 other compartments. Estrogen receptor alpha, on the other hand, was nearly exclusively expressed in the zona parenchymatosa and zona vasculosa of the stroma. Our results show that cells obtained after laser microdissection from FFPE ovarian material can be successfully used for subsequent real-time PCR, despite the fact the RNA quality indicator number of the isolated RNA was usually comparatively low. The data of our immunohistochemical analysis support the expression data of our RNA studies. In conclusion, laser-capture microdissection in combination with quantitative PCR is a reproducible and reliable technique for quantification of a small number of cells from FFPE material. We gratefully acknowledge the continuous support by the DFG-Graduiertenkolleg 1029 and the BMBF (ARG 08/013).

Loss of Nkx3.1 leads to the activation of discrete downstream target genes during prostate tumorigenesis

SUMMARYThe expression of NKX3.1, a transcriptional regulator and tumor suppressor gene in prostate cancer, is downregulated during early stages of prostate tumorigenesis. However, little is known of the alterations in gene expression that occur as a result of this event. We combined laser capture microdissection and gene expression profiling to analyze the molecular consequences of Nkx3.1 loss during prostate cancer initiation using Nkx3.1-deficient mice. This analysis identified a cohort of genes (loss-of-Nkx3.1 signature) that are aberrantly overexpressed during loss-of-Nkx3.1 driven tumor initiation. We studied the expression of these same genes in independent loss-of-Pten and c-myc overexpression prostate adenocarcinoma mouse models. Nkx3.1 expression is lost in prostate epithelial proliferation in both of these mouse models. However, Nkx3.1 loss is an early event of tumor development in the loss-of-Pten model, whereas it occurs at later stages in c-myc transgenic mice. A member of genes of the loss-of-Nkx3.1 signature, like clusterin and quiescin Q6, are highly expressed in prostatic hyperplasia and intraepithelial neoplasia (PIN) lesions that also lack Nkx3.1 in the Pten-deficient prostate, but not in similar lesions in the c-myc transgenic model. Meta-analysis of multiple prostate cancer gene expression datasets including those from loss-of-Nkx3.1, loss-of-Pten, c-myc overexpression, and constitutively active Akt prostate cancer models, further confirmed that genes associated with the loss-of-Nkx3.1 signature integrate with Pten-Akt signaling pathways, but do not overlap with molecular changes associated with the c-myc signaling pathway. In human prostate tissue samples, loss of NKX3.1 expression and corresponding clusterin overexpression are co-localized at sites of prostatic inflammatory atrophy, a possible very early stage of human prostate tumorigenesis. Collectively, these results suggest that the molecular consequences of NKX3.1 loss depend on the epithelial proliferative stage at which its expression is lost and, that alterations in the Pten-Akt-Nkx3.1 axis are important for prostate cancer initiation.

Appearance of epithelial and stromal genomic instability in background colorectal mucosa of sporadic colorectal cancer patients: relation to age and gender

We have previously demonstrated that not only epithelial but also stromal genetic instability possibly contributes to colorectal tumorigenesis. To assess the increasing risk of carcinogenesis in the colorectum with aging, we examined genomic instability in both epithelia and stroma in the background noncancerous mucosa of patients with colorectal carcinomas. In 213 noncancerous colorectal mucosa samples from colorectal cancer cases and 51 normal mucosa specimens of diverticulosis cases, epithelial and stromal genomic instability was analyzed with National Cancer Institute standard microsatellite markers, chromosome 17 (Chr.17) markers and tumor suppressor gene-related markers, using a combination of laser-capture microdissection and GeneScan approaches. Results were compared with immunohistochemically demonstrated expression of FHIT, Rb, WT1, hMLH1 and hMSH2. Genomic instability (MSI and LOH) in both epithelia and stroma appeared after around 40 years of age and remained relatively constant thereafter at relatively low frequencies (4.8-30.4%). The Epithelial LOH tended to show a stepwise increase in people in their 40s and 50s along with aging, especially in males. Overall frequencies of both epithelial MSI and LOH in left-side colon and LOH in right-side colon were significantly higher in males than in females. Epithelial hMLH1 expression in MSI (-) cases tended to be reduced with aging. Genomic instability of both MSI and LOH in noncancerous colonic mucosa, and more particularly epithelial and stromal LOH, appears relatively early in adults, suggesting age-related changes which increase the risk of cancer development, particularly in males.

Characterization of molecular markers indicative of cervical cancer progression

Cervical cancer originates with human papillomavirus (HPV) infection and progresses via histologically-defined premalignant stages. Here we compare normal cervical epithelium and patient-matched high grade squamous intraepithelial lesions (HSIL) with cervical carcinoma tissue from the same patient population (n=10 per group). Specimens were analyzed by combined laser capture microdissection and 2D-DIGE. Significant expression changes were seen with 53 spots resulting in identification of 23 unique proteins at the molecular level. These include eight that uniquely distinguish normal epithelium and HSIL and four that uniquely distinguish HSIL and carcinoma. In addition, one protein, cornulin, distinguishes all three states. Other identified proteins included differentiation markers, oncogene DJ-1, serpins, stress and interferon-responsive proteins, detoxifying enzymes, and serum transporters. A literature review, performed for all identified proteins, allowed most changes to be assigned to one of three causes: direct or indirect HPV oncoprotein interactions, growth selection during latency, or interactions in the lesion microenvironment. Selected findings were confirmed by immunohistochemistry using either frozen sections from the same cohort or formalin fixed paraffin embedded samples from a tissue microarray. Novel markers described here have potential applications for increasing the predictive value of current screening methods.

Gene expression profiling of the tumor microenvironment during breast cancer progression.

IntroductionThe importance of the tumor microenvironment in breast cancer has been increasingly recognized. Critical molecular changes in the tumor stroma accompanying cancer progression, however, remain largely unknown. We conducted a comparative analysis of global gene expression changes in the stromal and epithelial compartments during breast cancer progression from normal to preinvasive to invasive ductal carcinoma.MethodsWe combined laser capture microdissection and gene expression microarrays to analyze 14 patient-matched normal epithelium, normal stroma, tumor epithelium and tumor-associated stroma specimens. Differential gene expression and gene ontology analyses were performed.ResultsTumor-associated stroma undergoes extensive gene expression changes during cancer progression, to a similar extent as that seen in the malignant epithelium. Highly upregulated genes in the tumor-associated stroma include constituents of the extracellular matrix and matrix metalloproteases, and cell-cycle-related genes. Decreased expression of cytoplasmic ribosomal proteins and increased expression of mitochondrial ribosomal proteins were observed in both the tumor epithelium and the stroma. The transition from preinvasive to invasive growth was accompanied by increased expression of several matrix metalloproteases (MMP2, MMP11 and MMP14). Furthermore, as observed in malignant epithelium, a gene expression signature of histological tumor grade also exists in the stroma, with high-grade tumors associated with increased expression of genes involved in immune response.ConclusionsOur results suggest that the tumor microenvironment participates in tumorigenesis even before tumor cells invade into stroma, and that it may play important roles in the transition from preinvasive to invasive growth. The immune cells in the tumor stroma may be exploited by the malignant epithelial cells in high-grade tumors for aggressive invasive growth.

Quantitative cellular level analysis of mitochondrial DNA 3243A&gt;G mutations in individual tissues from the archival temporal bones of a MELAS patient

We could represent the first quantitative analysis of the mutation rate at the cellular level in human inner ear of a patient with MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) by combining laser capture microdissection (LCM) and quantitative real time PCR. We previously reported combining LCM and PCR to isolate mtDNA from the cells of specific tissues within a human archival celloidin-embedded temporal bone section without known otological history. Using this method, we quantitatively analyzed the rate of mtDNA 3243A > G mutation in the inner ear of a MELAS patient, and examined the correlation of the mutation rate at the cellular level and their histopathological condition. We extracted each inner ear organs using LCM from temporal bone sections of a MELAS patient, and studied the mutation rate, which was calculated as the ratio of the amount of mutant mtDNA to the total mtDNA. We found that the mtDNA mutation rate was high in spiral ganglion cells and the saccular macula, but was comparatively low in hair cells of the organ of Corti, the stria vascularis and the facial nerve. With the exception of the stria vascularis, there was a good correlation between the mutation rate and the histological findings.

Laser capture microdissection and cDNA array analysis for identification of mouse KIAA/FLJ genes differentially expressed in the embryonic dorsal spinal cord

During early development, centrally projecting dorsal root ganglion (DRG) neurons extend their axons toward the dorsal spinal cord. We previously reported that this projection is achieved by dorsal spinal cord-derived chemoattraction. However, the molecular nature of the chemotrophic cue is not yet fully understood. To identify novel genes differentially expressed in the dorsal spinal cord in the embryonic day 10.5 mouse, we used the Kazusa cDNA array system comprising approximately 1700 mouse KIAA/FLJ (mKIAA/mFLJ) cDNA clones and laser capture microdissection (LCM) in combination with PCR-based cDNA amplification. We observed that a certain population of genes showed significantly increased expression in the dorsal spinal cord. In situ hybridization analysis verified the expression of mRNAs of 6 genes ( Hip1r, Nav2, Fstl5, Cacna1h, Bcr, and Bmper) in the cells that constitute the dorsal spinal cord. The dorsal spinal cord-specific genes identified in this study provide a basis for studying the molecular nature of the neural development including the axonal guidance of DRG neurons. These results also demonstrate that the combined use of LCM coupled with the Kazusa cDNA array technology will be useful for the identification of large proteins expressed in the restricted small regions of embryos.

Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions.

The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling, which is decisive in cellular transcriptomic exploration. LCM makes possible the isolation of unique cells or group of cells, but maintaining RNA quality during this process is challenging. Several protocols are available for section preparation, but none of those guarantees the integrity of the RNA during microdissection, and operators are recommended to perform LCM during a limited time. We hypothesized that the cause of RNA degradation during the microdissection time is the presence of water rendering endogenous RNase activity possible. We thus developed two methods that stabilize RNA during microdissection time for up to 90 min. The first one consists of performing LCM under an argon atmosphere, thus preventing tissue rehydration; it is compliant with all existing microdissection protocols. The second one is a new fixation and staining method using ethanol as solvent in all preparatory steps to LCM that enhances fixation and dehydration of samples. We assessed several stains in regard of their effect on tissue morphology and RNA integrity and adjusted an ethanolic staining solution of cresyl violet and eosin Y.

Spatial Differences in an Integral Membrane Proteome Detected in Laser Capture Microdissected Samples

The combination of laser capture microdissection and mass spectrometry represents a powerful technology for studying spatially resolved proteomes. Moreover, the compositions of integral membrane proteomes have rarely been studied in a spatially resolved manner. In this study, ocular lens tissue was carefully dissected by laser capture microdissection and conditions for membrane protein enrichment, trypsin digestion, and mass spectrometry analysis were optimized. Proteomic analysis allowed the identification of 170 proteins, 136 of which were identified with more than one peptide match. Spatial differences in protein expression were observed between cortical and nuclear samples. In addition, the spatial distribution of post-translational modifications to lens membrane proteins, such as the lens major intrinsic protein AQP0, were investigated and regional differences were measured for AQP0 C-terminal phosphorylation and truncation.

Proteomic Analysis of Laser-Captured Paraffin-Embedded Tissues: A Molecular Portrait of Head and Neck Cancer Progression

Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalent cancer among men worldwide, is associated with poor prognosis, which has improved only marginally over the past three decades. A proteomic analysis of HNSCC lesions may help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC. Laser capture microdissection was combined with recently developed techniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and a novel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections of normal oral epithelium and well, moderately, and poorly differentiated HNSCC were processed for mass spectrometry and bioinformatic analysis. A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. Of interest, the relative expression of many of these molecules followed a distinct pattern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumor tissues. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays. The ability to combine laser capture microdissection and in-depth proteomic analysis of FFPE tissues provided a wealth of information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression, which may allow the development of novel biomarkers of diagnostic and prognostic value and the identification of novel targets for therapeutic intervention in HNSCC.

Quantitative Proteomic Profiling of Colorectal Cancer Stratified by Lymph Node and 8p LOH Status

Purpose: An objective of clinical proteomics, as applied to colorectal cancer (CRC), is to identify patients who are at greater risk of recurrence following initial resection and thus in need of more aggressive management. We applied sensitive proteomic technology to identify biomarkers that correlate with established prognostic indicators including lymph node status and LOH at chromosome 8p. Methods: We compared 48 tissue samples: 12 node-positive CRC, 12 node-negative CRC, and patient-matched normal mucosa. Laser capture microdissection (LCM) was used to isolate pure cell populations from standard frozen sections. About four micrograms of protein was analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). About 200,000 data features were quantified, compared and analyzed using a Significance Analysis of Microarrays (SAM) procedure, which provides a list of candidate biomarkers ranked by priority. Mass spectrometry for molecular identification of candidate biomarkers is in progress. Results: More than 2100 features per gel have been resolved. We project that about 1000 features will be matched as common across 90% or more of the gel images. Interim analysis of 604 well-matched features shows 111 with highly significant changes between cancer and normal (based on SAM analysis with estimated false discovery rate of < 1%). A heat map in Fig. 1 shows an overview of the interim analysis. In addition, as compared between node positive and node negative samples, at least one feature has been identified to date. Characteristics of this and other candidate biomarkers will be discussed.[figure1]FigureConclusion: The combination of LCM and 2D-DIGE provides a comprehensive approach for proteomic analysis of very small amounts of CRC tissue obtained from standard clinical specimens.

Clonal Analysis of Human Embryonic Stem Cell Differentiation into Teratomas

Differentiation of human embryonic stem cells (HESCs) can be studied in vivo through the induction of teratomas in immune-deficient mice. Cells within the teratomas differentiate into all three embryonic germ layers. However, the exact nature of the proliferation and differentiation of HESCs within the teratoma is not fully characterized, and it is not clear whether the differentiation is cell autonomous or affected by neighboring cells. Here, we establish a genetic approach to study the clonality of differentiation in teratomas using a mixture of HESC lines. We first demonstrate, by means of 5-bromo-2'-deoxyuridine incorporation, that cell proliferation occurs throughout the teratoma, and that there are no clusters of undifferentiated-proliferating cells. Using a combination of laser capture microdissection and DNA fingerprinting analysis, we show that different cell lines contribute mutually to the same distinctive tissue structures. Further support for the nonclonal differentiation within the teratoma was achieved by fluorescence in situ hybridization analysis of sex chromosomes. We therefore suggest that in vivo differentiation of HESCs is polyclonal and, thus, may not be cell autonomous, stressing the need for a three-dimensional growth in order to achieve complex differentiation of HESCs. Disclosure of potential conflicts of interest is found at the end of this article.

The TP53 and RPS6 alterations at the invasive tumor front, center and stroma of oral squamous cell carcinoma

To assess the difference of genetic alteration patterns among different areas in the same oral squamous cell carcinoma (OSCC). Studied the loss of heterozygosity (LOH) and microsatellite instability (MI) at chromosomal loci TP53 and RPS6 on the invasive tumor front (ITF), the center/superficial part and stroma cells by combining laser capture microdissection (LCM) and PCR technique. There existed a high frequency of LOH and MI on chromosomes loci TP53 and RPS6. The frequency of RPS6 and TP53 aberration at the stroma was 23.5% (4/17) and 43.8% (7/16), respectively. While in epithelial part (both ITF and center), it reached up to 64.7% (11/17) and 70.6% (12/17) respectively, and the difference was significant (P < 0.05). The overall frequency of the two markers was statistically higher at the ITF (20/32) than at the center/superficial part (15/34) (P < 0.05). The current study revealed that genetic alterations were different in different areas of the same tumor and there existed a relationship between the histological grading and genotypes of OSCC.

Disabled-2 Is Expressed in Adrenal Zona Glomerulosa and Is Involved in Aldosterone Secretion

The differentiation of the adrenal cortex into functionally specific zones is probably due to differential temporal gene expression during fetal growth, development, and adulthood. In our search for adrenal zona glomerulosa-specific genes, we found that Disabled-2 (Dab2) is expressed in the zona glomerulosa of the rat adrenal gland using a combination of laser capture microdissection, mRNA amplification, cDNA microarray hybridization, and real-time RT-PCR. Dab2 is an alternative spliced mitogen-regulated phosphoprotein with features of an adaptor protein and functions in signal transduction, endocytosis, and tissue morphogenesis during embryonic development. We performed further studies to analyze adrenal Dab2 localization, regulation, and role in aldosterone secretion. We found that Dab2 is expressed in the zona glomerulosa and zona intermedia of the rat adrenal cortex. Low-salt diet treatment increased Dab2-long isoform expression at the mRNA and protein level in the rat adrenal gland, whereas high-salt diet treatment did not cause any significant modification. Angiotensin II infusion caused a transient increase in both Dab2 isoform mRNAs in the rat adrenal gland. Dab2 overexpression in H295R human adrenocortical cells caused an increase in aldosterone synthase expression and up-regulated aldosterone secretion under angiotensin II-stimulated conditions. In conclusion, Dab2 is an adrenal gland zona glomerulosa- and intermedia-expressed gene that is regulated by aldosterone secretagogues such as low-salt diet or angiotensin II and is involved in aldosterone synthase expression and aldosterone secretion. Dab2 may therefore be a modulator of aldosterone secretion and be involved in mineralocorticoid secretion abnormalities.