Atlas Human cDNA Expression Array Research Articles

CDNA Expression Array Analysis of Gene Expression in Human Hepatocarcinoma Hep3B Cells Induced By <italic>BNIPL-1</italic>

Bcl-2/adenovirus E1B 19 kDa interacting protein 2 like-1 (BNIPL-1) is a novel human protein identified in our laboratory, which can interact with Bcl-2 and Cdc42GAP and induce apoptosis via the BNIP-2 and Cdc42GAP homology (BCH) domain. In the present study, we established the Hep3B-Tet-on stable cell line in which expression of BNIPL-1 can be induced by doxycycline. The cell proliferation activity assay showed that the overexpression of BNIPL-1 suppresses Hep3B cell growth in vitro. The differential expression profiles of 588 known genes from BNIPL-1-transfected Hep3B-Tet-on and vector control cells were determined using the Atlas human cDNA expression array. Fifteen genes were differentially expressed between these two cell lines, among which seven genes were up-regulated and eight genes were down-regulated by BINPL-1. Furthermore, the differential expression result was confirmed by semiquantitative RT-PCR. Among these differentially expressed genes, p16INK4, IL-12, TRAIL and the lymphotoxin beta gene involved in growth suppression or cell apoptosis were up-regulated, and PTEN involved in cell proliferation was down-regulated by BNIPL-1. These results suggest that BNIPL-1 might inhibit cell growth though cell cycle arrest and/or apoptotic cell death pathway(s).

Gene array identification of osteoclast genes: differential inhibition of osteoclastogenesis by cyclosporin A and granulocyte macrophage colony stimulating factor.

Treatment of adherent peripheral blood mononuclear cells (PBMCs) with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) stimulates the formation of multinucleate osteoclast-like cells. Treatment with M-CSF alone results in the formation of macrophage-like cells. Through the use of Atlas human cDNA expression arrays, genes regulated by RANKL were identified. Genes include numerous cytokines and cytokine receptors (RANTES and CSF2R proportional, variant ), transcription factors (nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and GA binding protein transcription factor alpha (GABPalpha)), and ribosomal proteins (60S L17 and 40S S20). Real-time PCR analysis showed significant correlation (R2 of 0.98 P < 0.01) with array data for all genes tested. Time courses showed differential activation patterns of transcription factors with early induction of FUSE binding protein 1 (FBP) and c-Jun, and later steady upregulation of NFATc1 and GABP by RANKL. Treatment with cyclosporin A, a known NFATc1 inhibitor, resulted in a blockade of osteoclast formation. The mononuclear cells resulting from high cyclosporin treatment (1,000 ng/ml) were cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) positive but expression of calcitonin receptor (CTR) was downregulated by more than 30-fold. Constant exposure of M-CSF- and RANKL-treated cells to GM-CSF resulted in inhibition of osteoclast formation and the downregulation of CTSK and TRAP implicating the upregulation of CSF2R in a possible feedback inhibition of osteoclastogenesis.

Differential gene expression in human hepatocellular carcinoma Hep3B cells induced by apoptosis-related gene BNIPL-2.

Bcl-2/adenovirus E1B 19 ku interacting protein 2-like (BNIPL-2) is a novel protein recently identified in our laboratory. BNIPL-2 is homologous to human BNIP-2, a potentially proapoptotic protein, and can interact with Bcl-2 and Cdc42GAP and promote apoptosis in BEL-7402 cells. Here we report the gene-expression profile regulated by BNIPL-2 in human hepatocarcinoma Hep3B cells and the analysis of its potential roles in cell apoptosis. BNIPL-2 was overexpressed in Hep3B cells using tetracycline inducible or Tet-on system. Screened by Western blot, the cells with low background and high induction fold of BNIPL-2 were obtained. We performed Atlas human cDNA expression array hybridization on these cells and analyzed the data with Quantarray software to identify BNIPL-2-regulated genes and their expression profile. RT-PCR was used to confirm the altered expression level of part of genes identified by the Atlas array hybridization. Fifteen of 588 genes spotted on the Atlas membrane showed altered expression levels in BNIPL-2-transfected Hep3B-Tet-on cells, in which 8 genes involved in cell apoptosis or growth inhibition were up-regulated and 7 genes involved in cellular proliferation were down-regulated following overexpression of BNIPL-2. cDNA array is a powerful tool to explore gene expression profiles under inducible conditions. The data obtained using the cDNA expression microarray technology indicates that BNIPL-2 may play its roles in apoptosis through regulating the expression of genes associated with cell apoptosis, growth inhibition and cell proliferation.

Expression patterns of cell cycle and apoptosis‐related genes in a multidrug‐resistant human colon carcinoma cell line

Background: An in vitro multidrug resistance (MDR) system from a human colonic cancer cell line (SW620‐MDR) has been established. To further study the mechanisms at molecular level and prevention of multidrug resistance in clinical practice, it was demonstrated that the expressions of several apoptosis‐related and cell cycle regulator genes were changed in the cells. Methods: A multidrug‐resistant colonic cell line (SW620‐MDR) was established, and the Atlas human cDNA expression array was used for studying the pattern of gene expression in this cell line. Furthermore, Northern hybridization or real‐time PCR analysis confirmed the pattern of gene expression. Results: In the SW620‐MDR cell line the pro‐apoptosis genes, CASP4, BIK, PDCD2, and TACE were expressed with decreased levels, and the anti‐apoptosis genes CD27‐L and IGFBP2 were over‐expressed. Furthermore, the cell cycle regulator genes such as CDK6, CCND1, CDC27HS, CDC16HS, Wee1Hu, MAPKK1, and IGFBP6 were expressed with decreased levels in the drug‐resistant cell line. Conclusions: It is worthwhile investigating whether the differentially expressed pattern of the aforementioned genes exists in the drug‐resistant cancer specimens, and to further understand their functions in the cancer drug‐resistance mechanism.

Altered expression of a cell-cycle suppressor gene, Tob-1, in endometriotic cells by cDNA array analyses

Objective: Interleukin (IL)-1β, a product of activated peritoneal macrophages, is a central cytokine coordinating neovascularization and monocyte chemotaxis in endometriotic implants. To evaluate the effects of this cytokine on normal endometrial stromal cells and endometriotic stromal cells we performed cDNA expression array analyses before and after exposure to IL-1β. Design: Nested case-control study of women with and without laparoscopic evidence of endometriosis. Setting: Reproductive endocrinology clinic at a university hospital. Patient(s): Endometriosis and normal endometrial biopsies from eight patients were used to prepare stromal cell cultures from which mRNA was extracted. Intervention(s): None. Main Outcome Measure(s): Commercially available expression arrays (Atlas Human cDNA Expression Array, Clontech, representing 597 individual genes) were used to screen for mRNAs whose expression was affected by 12 hours of exposure to IL-1β (10 ng/mL). Northern blotting and subsequent quantitative densitometric evaluation was done to confirm steady-state levels of Tob-1 mRNA transcripts. Result(s): Array analyses revealed a cell-cycle regulatory gene, Tob-1, which was differentially expressed by the two cell types after incubation with IL-1β. Tob-1 was reduced 48% in endometriotic stromal cells exposed to IL-1β, but there was only a 16% reduction in normal endometrial stromal cells. Replicate Northern analyses (n = 4) showed that exposure to IL-1β for 12 hours resulted in a 25% ± 5% diminution of Tob-1 mRNA in endometriotic stromal cells. In contrast, no significant decrease (<3%) was observed in IL-1β exposed normal endometrial stromal cells. Conclusion(s): Tob-1, a cell-cycle inhibitor gene is differentially responsive to IL-1β in endometriotic stromal cells compared to normal endometrial stromal cells. IL-1β down-regulated Tob-1 in endometriotic stromal cells, but had no significant effect on normal endometrial stromal cells. Our results suggest that IL-1β promotes growth of endometriotic lesions through inhibition of Tob-1. These findings are the first to associate IL-1β with an alteration of cell-cycle gene expression in cells derived from endometriotic implants.

Study of Gene Expression in Thyrotropin-Stimulated Thyroid Cells by cDNA Expression Array: ID3 Transcription Modulating Factor as an Early Response Protein and Tumor Marker in Thyroid Carcinomas

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with 32P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

Gene expression profile in oral squamous cell carcinomas and matching normal oral mucosal tissues from black Africans and white Caucasians: the case of the Sudan vs. Norway

Expression profile of 588 known genes relating to tumour biology, was examined between oral squamous cell carcinomas (OSCCs) and matching normal oral mucosal tissues (NOMTs) obtained from Sudanese ( n=11) and Norwegian ( n=11) patients. cDNA probes were synthesised from total RNA and hybridised with the Atlas human cancer cDNA expression array membranes. RT-PCR and immunohistochemistry were applied to confirm the expression pattern of a subset of the 588 genes. Differences in expression of the genes examined were found between the OSCCs and the NOMTs on the Atlas membranes. Several of these genes were either up- or down-regulated 1.6-fold or higher in the OSCCs compared to the NOMTs in the cases from the two populations. We found that 181 (31%) and 195 (33%) genes were either up-regulated or down-regulated in the OSCCs from the Sudan and Norway, respectively. From the total number of genes ( n=376) found expressed in the OSCCs investigated from the two countries, 53 genes (14%) showed common expression profile [35 (66%) were up-regulated and 18 (34%) were down-regulated] and 70 genes (19%) showed opposite regulation status. Results of the RT-PCR and immunohistochemistry confirmed the hybridisation data. These findings may provide an OSCCs-specific gene expression profile in patients from the two countries, suggesting that alterations of 123 genes are common in these OSCCs regardless of ethnic differences or other socio-cultural risk factors between the patients from the two countries. The findings might further suggest that specific genes are frequently involved in these OSCCs, which may provide novel clues as diagnostic, prognostic biomarkers and/or targets for therapy. The Atlas human cancer cDNA expression array technique can be useful to examine and describe the expression profile of known genes frequently involved in OSCCs from different populations.

Insulin-like growth factor binding protein-3 is upregulated in LPS-treated THP-1 cells.

Lipopolysaccharide (LPS) is a potent activator of human monocytic cells. We have determined that LPS stimulation of the human monocytic cell line, THP-1, results in an increased apoptotic rate. We hypothesized that cDNA expression array analysis could be used to identify target genes involved in the regulation of this process. THP-1 cells (1 x 10(6)/mL) were stimulated with LPS (1 microg/mL) or vehicle control. Apoptosis was measured at 0, 24, 48, 72 and 96 h using propidium iodide staining and flow cytometry to determine the percentage of cells with hypodiploid DNA. At 16 h, the Atlas Human cDNA expression array system, containing probes for 205 genes related to apoptosis, was used to survey and quantify transcript expression. The experiment was performed in duplicate and the membranes were normalized to cytoplasmic beta-actin. Standard Western blotting was performed on the conditioned medium to correlate secreted protein expression with RNA expression. Pretreatment with insulin-like growth factor I (IGF-I) was performed to determine whether the effects of insulin-like growth factor binding protein-3 (IGFBP-3) on apoptosis were IGF-dependent. LPS stimulation of THP-1 cells resulted in a greater than 2-fold increase in the rate of apoptosis when compared to vehicle control. When the cDNA expression arrays were compared, there was a 500-fold increase in the expression of the IGFBP-3 transcript in the LPS-stimulated cells. Western blotting of culture medium verified an approximately 2-fold increase in secreted IGFBP-3. Pretreatment with IGF-I did not prevent the increase in apoptosis seen with LPS stimulation. THP-1 cell apoptosis is increased in response to LPS stimulation and is associated with a significant induction of IGFBP-3 mRNA and protein. IGFBP-3, which reportedly promotes apoptosis and modulates the bioavailability of the pro-survival insulin-like growth factor 1, may serve to regulate apoptosis in monocytic cells in an IGF-independent manner. These data further support the investigation of the role of the IGF axis in programmed cell death of immune cells.

Activation of the nuclear transcription factor κB (NFκB) and differential gene expression in U87 glioma cells after exposure to the cytoprotector amifostine

Purpose : Amifostine has been approved as a therapy to decrease the incidence of moderate-to-severe xerostomia in patients undergoing postoperative radiation treatment for head-and-neck cancer. As a reducing agent capable of participating in intracellular reductive/oxidative processes, it has the potential to affect redox-sensitive transcription factors and gene expression. Amifostine’s active free thiol WR-1065 was investigated to determine its effect on nuclear transcription factor κB (NFκB) activation and subsequent gene expression in U87 glioma cells. Methods and Materials : The human glioma cell line U87 was grown to confluency and then exposed to WR-1065 at a concentration of 40 μM for times ranging from 30 min to 24 h. Changes in cell cycle were monitored by flow cytometry. The effect of WR-1065 on NFκB activation was determined by a gel shift assay. Changes in gene expression as a function of time of exposure to WR-1065 were determined by Northern blot and the Atlas Human cDNA Expression Array (Clontech, Palo Alto, CA). Changes in gene expression using the Atlas Array were verified by reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers. Results : Exposure of U87 cells to 40 μM WR-1065 resulted in a marked activation of NFκB between 30 min and 1 h after treatment. Expression of MnSOD, an NFκB-responsive gene, was enhanced by over 2-fold after 16 h of treatment and remained elevated at 24 h. During this period of time, no changes in cell cycle distribution were observed. To assess changes in the expression levels of NFκB-responsive genes as a function of WR-1065 exposure, cDNA arrays containing 49 genes identified as having DNA-binding motifs for NFκB were used. Only five genes were found to be significantly affected at 1, 4, and/or 16 h of treatment. GST-3 and c-myc were repressed up to 2- and 4-fold, respectively. The expression levels of IL-2Ra, RANTES, and c-myb, in contrast, were enhanced up to 14-, 3-, and 2-fold, respectively. The remaining genes having NFκB-responsive elements in their promoter regions were either not expressed (20 genes) or were not affected (24 genes) by exposure to WR-1065. Conclusions : The redox-sensitive transcription factor NFκB can be activated in U87 glioma cells by the active thiol form of the cytoprotector amifostine. Activation of NFκB by the antioxidant WR-1065 is accompanied by a reduced expression of the oncogene c-myc and an enhanced expression of the antioxidant gene MnSOD, a gene whose expression in tumor cells is relatively low, but when overexpressed has been correlated with a suppression of the malignant phenotype. Activation of NFκB by WR-1065, however, results in selective rather than global changes in the expression of genes containing NFκB-responsive elements.

Profiling of retinoid mediated gene expression in synchronized human SCC cells using Atlas human cDNA expression arrays.

While retinoids have been demonstrated to inhibit growth of many tumor cells, including SCC cells, the molecular mechanism by which retinoids suppress growth has not been elucidated. We previously found that the growth of SCC cells was significantly inhibited by all-trans-retinoic acid (all-trans-RA) treatment, and this inhibition was dependent on the binding and activation of RARs. These nuclear receptors bind retinoids and alter the rate of transcription of specific genes. To identify targets of the activated RARs which mediate growth inhibition, we growth arrested SCC-25 cells in G-0 and examined the effect of all-trans-RA on synchronized SCC-25 cells. All-trans-RA inhibited G-1 progression in quiescent SCC-25 cells stimulated by FBS. More specifically, we found that the all-trans-RA execution point maps to mid/late G-1, 6 to 10 h after stimulation. Using this synchronized cell system, we examined the expression of cell cycle regulatory genes in quiescent SCC-25 cells stimulated with FBS and treated with all-trans-RA. We found few changes in expression of these genes which could account for all-trans-RA inhibition of SCC-25 cell growth. In order to compare the patterns of expression of a wider selection of genes in all-trans-RA treated and non-treated SCC-25 cells, we have used expression array technology. We successfully performed expression profiling experiments on the Atlas Human cDNA arrays which contain 1176 human genes. We have identified several up-regulated and several down-regulated gene expression changes mediated by all-trans-RA treatment in synchronized SCC-25 cells. This novel information will be useful in defining the mechanism by which retinoids suppress the growth of SCC cells.

Differential Expression of Critical Cellular Genes in Human Lung Adenocarcinomas and Squamous Cell Carcinomas in Comparison to Normal Lung Tissues

The Atlas human cDNA expression array was used to evaluate gene expression profile changes in the genesis of human lung adenocarcinomas and squamous cell carcinomas. Gene expression changes between adenocarcinomas and squamous cell carcinomas were also analyzed. Of the 588 gene targets, 262 genes were expressed in these tissues and, of these, 45 genes were differentially expressed by at least two-fold in tumor tissues compared to corresponding normal tissues. Semiquantitative reverse-transcriptase polymerase chain reaction was used to confirm gene expression changes. Only those genes that reflected changes in >50% of the analyzed tissues were included in the final analysis. Ultimately, 26 genes were evaluated with 14 genes overexpressed and 12 genes underexpressed compared to matching normal lung tissues. Although similar expression changes were detected in adenocarcinomas and squamous cell carcinomas for most of the genes analyzed, some subtype-specific differences were also found. Genes encoding cell cycle regulators, intracellular signal transducers, cell receptor and adhesion molecules, growth factors, oncogenes, and apoptotic effectors were differentially expressed in this study. These gene expression changes may directly contribute to the initiation or progression of human lung cancer or may be secondary effects of the tumorigenesis process. Regardless, many of these differences may be useful in the diagnosis and/or treatment of this deadly disease.

Helicobacter pylori infection induced alteration of gene expression in human gastric cells

BACKGROUNDHelicobacter pylori, a human pathogen responsible for many digestive disorders, induces complex changes in patterns of gene expression in infected tissues. cDNA expression arrays provide a useful tool for studying...

Human cDNA expression arrays in normal and malignant monocytes

New technologies have recently become available with complementary DNA (cDNA) microarrays, which allow simultaneous analysis of multiple cell gene expression. In hematological malignancies few data have been reported so far. We decided to investigate the usefulness of this challenging methodology in combination with immunomagnetic cell separation techniques. For that purpose, we performed a series of consecutive microarrays on different cell populations. Our samples consisted of monocytes from healthy donors, AMML and CMML patients. As reference we used cells from the KBM3 monocytic leukemia cell line. Cell separations based both on negative and positive selection were tested, in order to verify possibility of gene activation or suppression elicited by the interaction of monoclonal antibodies and surface antigens. After density gradient-based mononuclear cell enrichment, CD14+ cells were sorted using the MACS recommended procedures. FACS analysis was performed on separation products to test the purity of monocytic populations. Both mRNA and total RNA were tested for some of the samples, 32P-radiolabeled cDNA was synthesized and hybridized to Atlas Human cDNA expression array (Clontech) encompassing 588 selected genes. We were able to identify some major recurrent differences between normal and CMML monocytes. Results obtained with mRNA and total RNA were comparable. Negatively-isolated CMML monocytes showed an overexpression of chemokine genes such as migration inhibitory factor-related proteins 8 (MRP8) and 14 (MRP14) and a downregulation of the interleukin-13 (IL-13) precursor gene. Interestingly, low level of expression of some of these genes appears to be upregulated by positive selection performed by CD14-conjugated microbeads in normal monocytes, while no changes were observed in negatively selected monocytes. While human cDNA expression arrays seem to represent a reliable tool for the study of gene expression profiles, we experience some variability between different patients. In conclusion, our results demonstrate that cell separation methodology has an impact in the expression of certain genes and must be taken into consideration. Besides a statistical evaluation of the results, strict controls are always recommended.

Identification of differentially expressed genes in nasopharyngeal carcinoma by means of the Atlas human cancer cDNA expression array.

To investigate genes of critical areas, including cell cycle/growth control, apoptosis, oncogene/tumor suppressors and growth factor/cytokines, that are differentially expressed in nasopharyngeal carcinoma. The Human Cancer cDNA Atlas, which contains 588 genes relating to tumor biology, was used to screen normal nasopharyngeal tissue, nasopharyngeal cancer (NPC). The reverse transcription/polymerase chain reaction was used to confirm the expression pattern of some genes identified by Atlas hybridization. The differentially expressed cell cycle/growth control regulators in NPC showed a stronger tendency toward cell proliferation with the up-regulation of cyclin D1, cyclin D2 etc. The expression pattern of apoptosis-related genes demonstrated the up-regulation of both anti-apoptotic factors such as the BCL-2-related protein A1, TRAF3, the inhibitor of apoptosis protein A1 (IAPI) and apoptotic pathway elements such as Fas/Apo-1, Apo-2 ligand etc. Among oncogenes/tumor suppressors, MDM2, STAT1 and STAT2 were found to be up-regulated in NPC. The expression profile of growth factors/cytokines showed the up-regulation of many growth-enhancing factors such as EGR1, tumor-derived growth factor 1, platelet-derived growth factor A chain etc. as well as Th1-type cytokines e.g. interleukin-1beta and interferons. A smaller number of genes were down-regulated in nasopharyngeal cancer, such as those encoding ERK1, Raf, secreted apoptosis-related protein 1, CD27BP, transforming growth factor beta2, pre-B-cell-stimulating factor homologue etc. The consistent tendency toward cell proliferation, the possibility of a stronger antiapoptotic force that operates on the normal apoptotic pathway, or the autocrine or paracrine growth factors may account for the development of NPC. Some genes are reported for the first time to have changed expression in nasopharyngeal carcinoma. The simple, quick, and high-throughput method of profiling gene expression by cDNA array hybridization provides us with a quick overview of key factors that may be involved in NPC, and may identify genes suitable for further study of carcinogenesis mechanism or targets for possible molecular diagnosis or therapy.

Bone morphogenetic protein 2 (BMP-2) induces sequential changes of Id gene expression in the breast cancer cell line MCF-7.

Bone morphogenetic proteins (BMPs) are involved in the development of various organs including the mammary gland. They are well-regulated and act in a time-, concentration- and cell-type-specific manner. We found that BMP-2 is expressed in primary breast tumor tissue samples and in breast cancer cell lines. Hybridization of labeled cDNA, obtained from the breast cancer cell line MCF-7, against the Atlas human cDNA expression array revealed differential gene expression depending on BMP-2 treatment. The most prominent changes were observed for the helix-loop-helix proteins Id-1, Id-2 and Id-3. Id-1 expression had increased severalfold after 4 h and was even higher after 24 h. Id-2 and Id-3 were more strongly induced after 4 h and showed no further significant change after 24 h. Analysis of cell-cycle distribution revealed a marked increase of the sub-G1 phase after 48 h in serum-deprived cells. In the presence of BMP-2 no change was observed over 48 h indicating that BMP-2 does not induce apoptosis. In addition, expression of caspase-3 was reduced in BMP-2-treated cells after 24 h. In summary, our results clearly indicate that BMP-2 is a susceptibility factor keeping the cells ready for the integration of various other signals for cell progression.

The use of large-scale cDNA analysis to profile differential gene expression in KYSE 410 human esophageal cancer cells after irradiation.

Atlas Human cDNA Expression Array, which includes 588 genes divided into functional classes, was used to study gene expression profiles in KYSE 410 human esophageal cancer cells irradiated in doses of 6 and 18 Gy. Considerable changes in the expression of a number of genes was found, including down-regulation of 3 cell cycle regulators and up-regulation of an apoptosis-related gene. Comparison of the expression profiles and identification of genes were performed with Atlas Vision 3.0 software.

Differential Gene Expression Pattern between Normal Human Trophoblast and Choriocarcinoma Cell Lines: Downregulation of Heat Shock Protein-27 in Choriocarcinoma in Vitro and in Vivo

Objective.Our purpose was to identify potential differences in gene expression between normal trophoblast and choriocarcinoma cells.Methods. The Atlas human cDNA expression array hybridization technique was used to study the gene expression pattern in normal trophoblast and choriocarcinoma cell lines. Furthermore, to confirm heat shock protein-27 (Hsp-27) expression data, reverse transcriptase-PCR (RT-PCR), Western blot, and immunohistochemical analyses were used in vitro with cell lines and in vivo with paraffin sections.Results. The expression of nine genes was strongly different comparing a normal trophoblast cell line with choriocarcinoma cells on the Atlas membranes. Compared to normal trophoblast cells, six genes were upregulated and three were downregulated in choriocarcinoma cells. Furthermore, the downregulation of Hsp-27 in choriocarcinoma cells was confirmed both in vitro with cell lines and in vivo with paraffin sections using RT-PCR, Western blot, and immunohistochemical techniques.Conclusion. cDNA expression array is a useful technique for identifying differentially expressed gene patterns in normal trophoblast and choriocarcinoma cells. The strong expression of Hsp-27 in placental villous trophoblast cells may play a role in trophoblast differentiation. The downregulation of Hsp-27 in choriocarcinoma may contribute to the extreme sensitivity of trophoblastic tumors to chemotherapy.

CXCR-4, a chemokine receptor, is overexpressed in and required for proliferation of glioblastoma tumor cells.

Using the technique of differential hybridization of Atlas Human cDNA expression arrays, we previously reported the isolation of a G protein coupled receptor, CXCR-4, which is overexpressed in glioblastoma multiforme tumor tissue (GMTT) compared to normal brain tissue (NBT). Using gene specific reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization, we studied its expression in a variety of brain and breast tumor tissue samples. To demonstrate the requirement of CXCR-4 in glioblastoma cell proliferation an antisense construct was overexpressed. Glioblastoma cells were also treated with antibodies against CXCR-4 and its ligand, SDFbeta-1. Expression analysis indicated that CXCR-4 is overexpressed in 57% of the primary glioblastoma tissues and in 88% of the glioblastoma cell lines analyzed. Overexpression of CXCR-4 in glioblastoma cell lines enhanced their soft agar colony-forming capability. Expression of anti-sense CXCR-4 in glioblastoma cell lines caused neurite outgrowth and cellular differentiation. Treatment of glioblastoma cell lines with CXCR-4 and SDFbeta-1 specific antibodies caused inhibition of glioblastoma cell proliferation. On the basis of these results, we conclude that CXCR-4 gene is required for the proliferation of human glioblastoma tumors.

Application of the differential hybridization of Atlas Human expression arrays technique in the identification of differentially expressed genes in human glioblastoma multiforme tumor tissue.

Several molecular biology techniques are utilized to study changes in gene expression during the genesis of human tumors. Our objective was to identify genes that showed altered expression between normal brain tissue (NBT) and glioblastoma multiforme tumor tissue (GMTT). The technique of differential hybridization of two Atlas Human cDNA expression array was used. In this technique, dCTP32-labeled complimentary DNA from NBT and GMTT was hybridized to two identical human cDNA expression array membranes containing 588 known genes. Autoradiographic analysis showed that of the 588 genes analyzed, 52 are overexpressed in GMTT and 57 in NBT. A gene-specific semiquantitative reverse transcription polymerase chain reaction (RT-PCR) method was used to confirm the expression pattern of seven known genes. RT-PCR results demonstrate that the expression pattern of a majority of genes agreed with the expression pattern observed on expression array. The known tumor suppressor genes retinoblastoma (RB) and p53 showed loss of expression in GMTT compared with NBT. We conclude that the differential hybridization technique of Atlas Human cDNA expression array can be a useful method in identifying genes that are differentially expressed either in NBT or GMTT.