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Results for “mRNA overexpression”

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At least 19 recordsLinked to original sources

PubMed1992

Evaluation of epidermal growth factor receptor DNA amplification and mRNA expression in bladder cancer.

The epidermal growth factor receptor has been implicated in the malignant transformation of cells because the v-erbB oncogene is a truncated form of the epidermal growth factor receptor. DNA amplification and/or mRNA overexpression of the epidermal growth factor receptor is associated with the malignant potential of several human epithelial tumors. To determine the frequency of epidermal growth factor receptor DNA amplification and mRNA overexpression in bladder cancer, we evaluated 12 bladder tumors for DNA amplification and another 14 bladder tumors for mRNA overexpression. By Southern hybridization, we found no evidence of DNA amplification or gene rearrangements in 12 bladder tumors. Five of 14 bladder tumors overexpressed epidermal growth factor receptor mRNA four to 15 fold compared to normal urothelium from the same bladder by Northern analysis. These findings suggest that epidermal growth factor receptor amplification and/or gene rearrangement occurs infrequently in bladder cancer. Epidermal growth factor receptor mRNA overexpression, however, is found in 36% of bladder tumors and may play a role in bladder tumorogenesis.

Blotting, Northern↗

PubMed1992

Cytokine dysregulation in AIDS: in vivo overexpression of mRNA of tumor necrosis factor-alpha and its correlation with that of the inflammatory cytokine GRO.

The human immunodeficiency virus establishes an intimate interaction with the immune system. The virus can use cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (Il-1), to regulate its own expression by modifying the normal immunoregulatory network. We demonstrate that mRNA of the cytokine TNF-alpha from peripheral blood mononuclear cells is overexpressed in virtually all patients with AIDS who do not have active opportunistic infections compared with uninfected volunteers (p < 0.0001). This overexpression correlates with elevated mRNA levels of the recently discovered GRO (p < 0.05), a cytokine involved in the inflammatory response.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Beta 2-adrenergic receptor mRNA is overexpressed in neoplastic human thyroid tissues.

The expression of beta 2-adrenergic receptor (AR) mRNA was investigated in normal and neoplastic human thyroid tissues. A combination of techniques for reverse transcribing mRNA into cDNA and the incorporation of 32P-gamma ATP into the polymerase chain reaction (PCR)-generated fragments allowed us to detect beta 2-AR mRNA in surgically excised thyroid specimens. The levels of beta 2-AR cDNA generated by PCR in thyroid adenomas and cancers were 3.3 and 6.9 times, respectively, as high as that of normal thyroid tissues. These findings suggest that the level of beta 2-AR mRNA is correlated with the extent of differentiation in neoplastic tissues. The present study provides new insights into the relationships between the AR-adenylate cyclase system and the regulation of the growth and differentiation in neoplastic human thyroid tissues.

Base Sequence↗

PubMed1991

Cellular retinoic acid-binding protein type 2 mRNA is overexpressed in human psoriatic skin as shown by in situ hybridization.

In situ hybridization with full length mouse cellular retinoic acid-binding protein type 1 and cellular retinoic acid-binding protein type 2 cDNA derived RNA probes showed overexpression of cellular retinoic acid-binding protein type 2 mRNA in lesional hyperplastic psoriatic skin whereas cellular retinoic acid-binding protein type 1 mRNA was undetectable. This suggests that the previously reported increase of cellular retinoic acid-binding protein in psoriatic epidermis corresponds to increased translation of cellular retinoic acid-binding protein type 2 gene. Cellular retinoic acid-binding protein types 1 and 2 mRNAs were not detectable in normal epidermis; however, type 2 message was detected in non-hyperplastic, non-lesional skin of psoriatic patients thus before altered epidermal differentiation and hyperplasia are morphologically detectable.

Antisense Elements (Genetics)↗

PubMed2026

Application of a Translational Research Platform to Unveil Efficacy Signals and Mechanisms of Resistance of FGFR Inhibitors in Multiple FGFR-Altered Solid Tumors.

PURPOSE: The predictive value of fibroblast growth factor receptor (FGFR) amplifications (amp) and the role of FGFR mutations (mut) beyond known activating variants remain unclear. We aimed to establish a translational research platform to characterize FGFR alterations (alt) and explore their potential as predictive biomarkers for FGFR-targeted agents. EXPERIMENTAL DESIGN: This ambispective study included a retrospective analysis of patients with FGFR-alt tumors treated with selective FGFR inhibitors (FGFRi) and a prospective collection of longitudinal tumor samples. Patient-derived xenografts (PDX) were generated to investigate FGFRi mechanisms of action and resistance. Molecular characterization included genomic, transcriptomic, proteomic, and functional analyses using the Functional Annotation for Cancer Treatment (FACT) assay. RESULTS: Among 36 retrospectively analyzed patients, clinical benefit from FGFRis was observed in cases with FGFR mRNA overexpression or FGFR2/11q co-amp, but no association was found with the amplification levels. In archival tumor samples, exploratory proteomic analysis showed FGFR1-4 protein expression in 78% of FGFR1/2-amp tumors detected by fluorescence in situ hybridization. RNA sequencing identified a higher prevalence of FGFR mRNA overexpression than proteomic analysis. Among patients harboring FGFR-mut, only one bladder cancer with an FGFR3-mut S249C derived benefit. FACT assay supported the functional activity of selected variants, including FGFR3 T689M, and suggested potential resistance mechanisms involving PI3K/PTEN and MAPK pathway co-alterations. A prospective FGFR-alt PDX biorepository enabled exploratory biomarker analyses, supporting the hypothesis that FGFR1-4 mRNA expression may better reflect FGFR dependency than genomic alterations alone. CONCLUSIONS: These findings highlight the complexity of FGFR-driven oncogenesis and support integrative molecular approaches to refine patient selection for FGFR-targeted therapies.

Humans↗

PubMed1992

5-HT2 receptor mRNA is overexpressed in cultured rat aortic smooth muscle cells relative to normal aorta.

Proliferation of smooth muscle cells in arteries is associated with contractile hypersensitivity to serotonin (5-HT). A possible explanation is that smooth muscle cells express increased numbers of phospholipase C (PLC)-coupled 5-HT receptors (5-HTR), which could mediate contractile and mitogenic signals via phosphatidylinositol turnover. To test this hypothesis, we performed a molecular characterization of 5-HTR subtypes in normal aorta and passaged rat aortic smooth muscle cells (RASM) in culture. Northern blot analysis revealed that growth-arrested cultured cells expressed 5-HT2R mRNA at 50-fold greater levels than aorta. 5-HT1CR mRNA was not detected in either case. 5-HT stimulated intracellular Ca2+ mobilization (fivefold peak increase) and c-fos mRNA induction (10-fold peak increase); both responses were strongly inhibited by selective 5-HT2R antagonists. Specific agonists for the 5-HT1AR, 5-HT1BR, and 5-HT1DR failed to induce c-fos mRNA. Although 5-HT (10 microM) increased [3H]thymidine incorporation (28% relative to 10% calf serum), it was a weak mitogen for cultured RASM based on cell counts. Thus there is high level expression of 5-HT2R mRNA by cultured RASM relative to aorta, and the 5-HT2R appears to be the only 5-HTR subtype mediating early growth signals in these cells. These data suggest that, following arterial injury in vivo, smooth muscle cells may overexpress the 5-HT2R, resulting in 5-HT contractile hypersensitivity and increased responsiveness to other growth factors.

Animals↗

PubMed2026

A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.

Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.

Animals↗

PubMed1991

Expression of c-myc in cutaneous lymphomas and pseudolymphomas.

The expression of the proto-oncogene c-myc was studied in tumorous skin lesions of (cutaneous) lymphoproliferative diseases (3 cases of pseudolymphoma, 8 cases of non-Hodgkin lymphoma and 1 case of lymph node involvement in mycosis fungoides) in a total of 12 patients. c-myc mRNA levels were quantified by Northern blot analysis followed by densitometric evaluation of the specific bands. Higher levels of c-myc mRNA expression were observed in lymphomas as compared with pseudolymphomas (p greater than 0.05). No significant differences in c-myc mRNA values were detected between B and T cell pseudolymphomas. Our results suggest that c-myc mRNA overexpression is associated with malignant lymphomas of the skin. We conclude that the measurement of c-myc mRNA levels may contribute to further characterize cutaneous lymphoproliferative diseases.

Blotting, Northern↗

PubMedDate not supplied

Immunohistochemistry, in situ hybridization and polymerase chain reaction (PCR) in detecting c-myc expression in human malignancies.

The assessment of oncogene expression at cellular level is important in understanding the role of those genes in carcinogenesis. Using in situ hybridization and immunohistochemistry, the expression of oncogenes can be visualized in topographic relation to tissue morphology. In the present study, c-myc overexpression was studied in ten carcinomas of different origin (6 mammary adenocarcinomas, 2 vulvar and 2 bronchial squamous cell carcinomas) by in situ hybridization (ISH) with 35S-labeled RNA probes and by immunohistochemistry (IHC). DNA amplification and transcription of c-myc oncogene were also studied with polymerase chain reaction (PCR) using beta-globin as an intrinsic standard for DNA amplification. The effect of formalin fixation of c-myc expression was simultaneously studied. Half of the tumours (5/10) demonstrated c-myc mRNA overexpression by ISH performed on frozen sections and two of the samples were shown to over-express c-myc protein by IHC. Only two samples fixed in formalin showed positive signals for c-myc mRNA. None of the biopsies showed DNA amplifications either with ISH or PCR. The present results suggest that ISH with RNA probes is a useful method for detecting the transcription of activated oncogenes in malignant tissues, especially when applied on frozen sections. The results also indicate that in some cases, c-myc gene may be adequately transcribed to mRNA but the latter is not translated to the appropriate oncoprotein.

Adenocarcinoma↗

PubMed1992

Response of human breast cancer cells to heat shock and chemotherapeutic drugs.

Previous studies have shown that certain chemotherapeutic drugs are less effective on tumor cells when cells have been previously exposed to hyperthermia. In the present study, we have evaluated whether specific modifications in heat shock protein (hsp) expression are associated with resistance to anticancer drugs. RNA levels for hsp90, hsp70, and hsp27 were studied by Northern and slot blots, while proteins were studied by two-dimensional gel electrophoresis, in MCF-7/BK and MDA-MB-231 breast cancer cells. The sensitivities of these cells to doxorubicin, colchicine, 5-fluorouracil, cisplatin, actinomycin D, and methotrexate were tested by clonogenic assays. These techniques were applied to both cell lines before (control) and after heat shock. The study revealed that elevated hsp70 and hsp27 levels were associated with doxorubicin resistance. In addition, the presence of phosphorylated hsp27 isoforms was also associated with doxorubicin resistance. The study showed that elevated hsps were not associated with multidrug resistance. Heat shock did not induce P170 glycoprotein mRNA overexpression or resistance to the other drugs tested. We also found that the level of doxorubicin protection conferred by the overexpression of hsp was lower than that obtained in cells expressing a multidrug resistance phenotype (MDA-A1R cells). In these cells, heat shock did not confer additional doxorubicin resistance and hsp27 phosphorylation was deficient. Our studies suggest that specific hsps are associated with doxorubicin resistance in certain human breast cancer cells and that this mechanism seems to be independent of the multidrug resistance system.

Breast Neoplasms↗

PubMed1991

A phenotype conferring selective resistance to lipophilic antifolates in Chinese hamster ovary cells.

Trimetrexate, a lipid-soluble analogue of methotrexate, appears to enter mammalian cells by passive diffusion, thus circumventing the methotrexate transport system which is frequently a subject for alterations leading to methotrexate resistance. Using a single-step selection protocol with trimetrexate, we have isolated 45 clonal variants and found the majority of them to be selectively resistant to lipophilic antifolates while retaining their sensitivity to methotrexate and drugs involved in multidrug resistance. The majority of spontaneously induced trimetrexate-resistant clones showed a change in neither the mRNA levels of dihydrofolate reductase (24 of 30) and P-glycoprotein (26 of 30) nor their gene copy numbers, whereas a small fraction of clones (4 of 30) showed multidrug resistance gene amplification and P-glycoprotein mRNA overexpression. gamma-Irradiation prior to selection markedly enhanced the frequency of trimetrexate resistance (100-fold after 1000 rads). None of the gamma-ray-induced trimetrexate-resistant clones (0 of 15) had evidence of dihydrofolate reductase and multidrug resistance gene amplification and/or overexpression. Flow cytometry data on trimetrexate-resistant clones showed no defect in the transport of trimetrexate. Verapamil, a modulator of the multidrug resistance phenotype, had no cytotoxic effect on parental and trimetrexate-resistant clones. However, when present with trimetrexate, verapamil (0.3-0.6 microM) reversed the lipophilic antifolate-resistant phenotype in clones that had invariant levels of P-glycoprotein and dihydrofolate reductase. This selective resistance to lipid-soluble antifolates was initially unstable but became stable after continued drug-selective growth. Two-dimensional gel electrophoresis showed some differences in protein(s) that may potentially be associated with this phenotype of selective resistance to lipophilic antifolates. We conclude that a gamma-radiation-enhanceable, verapamil-reversible, stable phenotype of selective resistance to lipid-soluble antifolates frequently emerges which requires neither the amplification nor the overexpression of dihydrofolate reductase or multidrug resistance genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Neu oncogene expression in ovarian tumors: a quantitative study.

We studied neu mRNA expression by slot blot analysis and protein product expression by capture ELISA and immunohistochemistry in 57 primary and metastatic ovarian neoplasms, two paraovarian leiomyosarcomas, and eight normal ovaries. Some 61% of ovarian tumors but none of the paraovarian neoplasms or normal ovaries overexpressed neu mRNA. A total of 96% of the ovarian tumors that overexpressed neu were of epithelial type. Epithelial ovarian tumors had significantly higher amounts of the neu oncogene product as determined by capture ELISA than either germ cell and stromal tumors or normal ovaries (p less than 0.025). Different subtypes of ovarian carcinomas had significantly different amounts of neu oncogene product as measured by capture ELISA; endometrioid tumors had the highest, and poorly differentiated carcinomas not otherwise specified had the lowest (p less than 0.025). ELISA values, mRNA overexpression, and immunohistochemical staining intensity did not correlate with stage at diagnosis or architectural or nuclear grade in ovarian tumors. We conclude that capture ELISA is a simple, effective way to measure the neu oncogene protein product and that there is a good correlation between ELISA levels and immunohistochemical staining intensity. However, ELISA values did not correlate with stage or histologic prognostic factors in ovarian neoplasms.

Carcinoma↗

PubMed1992

Expression of c-erbB2, TGF-beta 1 and pS2 genes in primary human breast cancers.

The presence of c-erbB2, TGF-beta 1 and pS2 mRNAs was examined in primary breast tumours. The c-erbB2 mRNA was overexpressed in 34% of the tumours. There was a positive, statistically significant correlation between c-erbB2 gene overexpression and nodal status. TGF-beta 1 mRNA was detected in 84% of the tumours, regardless of their clinical status. When possible, the c-erbB2 and TGF-beta 1 proteins were identified immunohistochemically on frozen sections from the same tumours. For TGF-beta 1, the mRNA and immunohistochemical results were divergent in 6 cases, 5 of which did contain clearly detectable mRNA but did not stain with the antibody. The pS2 mRNA was detected in 22% of the tumours and in the BT474 cell line. There was a significant correlation between the presence of pS2 mRNA and of oestrogen receptors. No statistically significant correlation was observed between pS2 and TGF-beta 1 genes expression and the clinical parameters of the tumours.

Aged↗

PubMed1992

Localization of overexpressed c-myc mRNA in polycystic kidneys of the cpk mouse.

The C57BL/6J-cpk mouse has a form of autosomal-recessive polycystic kidney disease characterized by the rapid growth of large collecting duct cysts and the development of severe renal failure usually by three to four weeks of age. Previous studies had shown higher steady-state levels of proto-oncogene mRNA in these cystic kidneys. It is now shown using nuclear run-on transcription that the c-fos and c-myc proto-oncogenes are transcribed at higher rates in cystic kidneys, and thus that increased transcription, in part, may account for the increased mRNA levels. c-myc mRNA was detected by in situ hybridization in nephron anlagen and elongating tubules of normal and cystic kidneys during late fetal and early neonatal kidney development. Localization of c-myc expression in the normal kidney decreased with age over the three-week postnatal period. By contrast, c-myc mRNA was found in cysts as early as three days of age, with increased levels at two and three weeks. c-myc expression was also elevated in apparently normal, non-dividing proximal tubules in three-week-old cystic animals. On the basis of these findings, we suggest that c-myc expression is linked to the proliferation of cells engaged in the primary cystogenic process, and that expression of this gene in proximal tubule cells of severely azotemic animals reflects the compensatory response of residual tubular epithelial cells to progressive renal dysfunction.

Animals↗

PubMedDate not supplied

Molecular analysis of two human doxorubicin-resistant cell lines: evidence for differing multidrug resistance mechanisms.

The molecular characteristics of two human doxorubicin-resistant cell lines were examined specifically for MDR1 gene amplification by Southern analysis and for overexpression of its messenger RNA. The 285-fold doxorubicin-resistant colon adenocarcinoma subline, LoVo/DR5, was found to overexpress the mRNA for P-glycoprotein without the concomitant requirement of MDR1 gene amplification, suggesting that relatively high levels of P-glycoprotein mediated multiple drug resistance may occur by transcriptional activation of the gene. Despite a similar in vitro selection strategy and in contrast to LoVo/DR cells, the 220-fold doxorubicin-resistant fibrosarcoma subline, HT1080/DR4, did not overexpress P-glycoprotein mRNA nor was the MDR1 gene amplified. In-gel renaturation studies were performed to determine the nature of a putative HSR-bearing chromosome 7 found in HT1080/DR4 cells; however, at a level of sensitivity nearing 20 copies of an amplified DNA fragment per haploid genome, no amplified sequences could be detected. These results suggest that doxorubicin resistance is multifactorial and alternative mechanisms of multiple drug resistance remain to be determined. LoVo/DR5 cells should prove to be a useful model for investigating transcriptional activation of the MDR1 gene; HT1080/DR4 cells should be an excellent model for the study of non-P-glycoprotein mediated multiple drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Effects of STZ-induced diabetes and fasting on insulin receptor mRNA expression and insulin receptor gene transcription in rat liver.

Insulinopenic states in rodents are known to cause an increase in the number of hepatic insulin receptors. To determine if this change is related to an abnormality in insulin receptor gene expression, insulin receptor binding, insulin receptor mRNA levels, and insulin receptor gene transcription rates have been measured in livers from rats rendered hypoinsulinemic by STZ administration (65 mg/kg) or fasting. In the two groups of experimental rats, insulin binding to liver plasma membranes was increased (approximately 40 and 25%, respectively) relative to control, normoinsulinemic animals. Northern blot analysis of either total or poly (A)+ RNA from livers of hypo- and normoinsulinemic rats revealed two major insulin receptor mRNA species of 9.5 and 7.5 kbs. In hypoinsulinemic rats, insulin receptor mRNA levels were increased > or = 10-fold, with similar effects on the two mRNA species. The effects of STZ administration and fasting on insulin receptor binding and insulin receptor mRNA levels were fully reversed by insulin treatment or refeeding, respectively. Injection of ACT D, an inhibitor of gene transcription, decreased insulin receptor mRNA levels by > or = 80% in control and diabetic rats and suppressed the overexpression of mRNA seen in diabetic rats. In vitro nuclear transcription assays showed that the rate of transcription of the insulin receptor gene was increased 2-fold in STZ-induced diabetic rats and fasted rats relative to control animals. Taken together, these results suggest that the upregulation of the insulin receptor induced by chronic insulinopenia results, at least in part, from an increase in insulin receptor gene transcription.

Analysis of Variance↗

PubMed1992

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.12.) gene expression in two malignant human mammary epithelial cell lines: BT-20 and MCF-7. Regulation of gene expression by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the control of glycolysis. Its gene expression was analyzed in two breast cancer cell lines of human origin, BT-20 and MCF-7. We used a cDNA probe of 1.3 kb for Northern blot hybridization. It is found that GAPDH mRNA is overexpressed only in the poorly differentiated BT-20 cell line and that treatment of these cells by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) stimulates GAPDH mRNA expression in a dose-and time-dependent manner. The present investigation on the BT-20 cells indicates that the expression of GAPDH is sensitive to 1,25-(OH)2D3 and up-regulated by low doses of this steroid.

Breast Neoplasms↗

PubMed1992

Reduced topoisomerase II and elevated alpha class glutathione S-transferase expression in a multidrug resistant CHO cell line highly cross-resistant to mitomycin C.

We have isolated a multidrug-resistant derivative of Chinese hamster ovary CHO-K1 cells by exposure to progressively increasing concentrations of Adriamycin. This cell line, designated CHO-Adrr, was 27-fold more resistant than the parental line to Adriamycin and showed similar degrees of cross-resistance to several other topoisomerase II (topo II) inhibitors, including mitoxantrone, daunomycin and etoposide. CHO-Adrr cells showed a lower (4-fold) level of cross-resistance to vincristine and colchicine, drugs associated with the multidrug-resistant phenotype. While CHO-Adrr cells showed no enhanced resistance to several mono- and bi-functional alkylating agents or to UV and ionizing radiation, they were greater than 80-fold resistant to mitomycin C (MMC). There was a 5-fold decreased level of daunomycin accumulation in CHO-Adrr cells compared to CHO-K1 cells and this was associated with increased drug efflux. The resistant cells had amplified multidrug resistance gene (mdr) sequences and overexpressed (mdr) mRNA. Verapamil was able to completely reverse Adriamycin resistance but reversal of MMC resistance was only partial, with residual 23-fold resistance. CHO-Adrr cells expressed a 4-fold reduced level of topo II protein but overexpressed an alpha class (basic) glutathione S-transferase (GST). Analysis of cell hybrids showed that while the level of resistance to Adriamycin dropped by a factor of 3 in CHO-K1/CHO-Adrr hybrids compared to CHO-Adrr/CHO-Adrr hybrids, resistance to MMC dropped 10-fold. Thus, CHO-Adrr cells appear to exhibit simultaneously several different drug resistance mechanisms including MDR and GST overexpression, and topo II reduction.

Amsacrine↗