A general method for isolation of individual poly-ribosomes and pure mRNAs by immunosorption technique.
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Animals↗
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Results for “RNA, Neoplasm”
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Studies of the UV-sensitivity of virus-specific RNA synthesis in cells infected or transformed by adenovirus 5 and SV 40.
The effect of UV-irradiation on host and virus-specific RNA synthesis in cells infected or transformed by tumor viruses (Ad 5 and SV 40) was studied. It was found that the synthesis of host and Ad 5 RNA in infected KB cells was almost euqally inhibited upon UV-irradiation; the transcription of the EcoRI produced fragements--fragment B was inhibited to a greater extent than fragment A, suggesting that the transcription of the whole Ad 5 genome starts from the left side. The transcription of viral sequences in transformed cells, on the other hand, was more resistant to UV-irradiation than the transcription of the host ones. The results obtained suggest that the small size of the virus transcriptions, or their location at the beginning of large host transcriptions, may be responsible for the observed data.
Adenoviridae↗
Reduced levels of topoisomerase II alpha and II beta in a multidrug-resistant lung-cancer cell line.
We have previously shown that the doxorubicin-selected multidrug-resistant small-cell lung-cancer cell line H69AR is resistant to VP-16-induced single-strand DNA breaks as compared with its parental H69 cell line. Levels of immunoreactive topoisomerase II alpha are also reduced in H69AR cells. In the present study, we found that cleaved complex formation in the presence of VP-16 was decreased in H69AR cells as compared with H69 cells. In addition, the resistant cells contained lower levels of both topoisomerase II alpha and topoisomerase II beta protein and mRNA. However, these changes were not accompanied by a decrease in the P4-unknotting (strand-passing) activity of 0.67 M NaCl nuclear extracts of H69AR cells, nor was there any difference in VP-16 inhibition of unknotting activity in the H69 and H69AR nuclear extracts. These data suggest that reduced levels of topoisomerase II alpha and II beta may contribute to the resistance of H69AR cells to VP-16 and other drugs that target these isoenzymes.
Blotting, Northern↗
Regulation of HSP60 mRNA expression in a human ovarian carcinoma cell line.
The expression of the 60-kDa heat-shock protein (HSP60) varies markedly among patients with ovarian carcinoma, and high-level expression predicts poor survival in such patients treated with cisplatin (DDP)-containing chemotherapy programs. We investigated the expression of HSP60 in human ovarian carcinoma 2008 cells and an 11-fold DDP-resistant subline 2008/C13*5.25. Heating for 2 h at 44 degrees C produced a 2.7 +/- 0.16-fold increase (mean +/- SD) that was maximal at 4 h after the start of heat exposure. Exposure to an IC50 concentration of DDP for 1 h induced a 1.8 +/- 0.03-fold increase in hsp60 expression. The opposite was true for cadmium and zinc, both of which induced increases in metallothionein IIA but not in the hsp60 message. 2008/C13*5.25 cells constitutively over-expressed hsp60 mRNA by 1.7 +/- 0.16 orders of magnitude and contained a 3.8 +/- 0.45-fold higher level of HSP60 as detected by immunocytochemical staining. 2008/C13*5.25 cells showed 1.2-fold cross-resistance to thermal killing. Expression of hsp60 was markedly reduced in 2008 xenografts as compared with 2008 cells growing in vitro; however, neither serum starvation nor refeeding altered the message level. Exposure to a variety of growth factors and drug treatments known to alter the DDP sensitivity of 2008 cells, including epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, buthionine sulfoximine, ouabain, and forskolin, did not alter hsp60 expression. These results suggest a role for HSP60 in mediating resistance to both DDP and hyperthermia but indicate that the hsp60 mRNA levels are not regulated by the factors listed above.
Blotting, Northern↗
Localization of host poly(A)+ mRNA in the ribosome profile of mengovirus-infected Ehrlich ascites tumor cells.
The distribution of poly(A)+ mRNA among polysomes, monosomes, and ribosome-free supernatant fractions after mengovirus infection of Ehrlich ascites tumor (EAT) cells was investigated employing sucrose gradient centrifugation of their corresponding postnuclear supernatants. Poly(A)+ mRNA was isolated from sucrose gradient fractions and quantitated in a cell-free protein synthesizing system from uninfected EAT cells. It was also localized by annealing [3H]-poly(U) to the poly(A)-tracts of mRNA present in the sucrose gradient fractions. Both experiments revealed a gradual shift of host poly(A)+ mRNA from large to small polysomes and monosomes, respectively, with the time postinfection. The greatest part of host template RNA appears to remain ribosome-bound and only a fraction seems to be detached from the ribosomes in the course of mengovirus infection. At the end of the infectious cycle, 8 h postinfection, approximately 70% of the poly(A)+ mRNA detected in uninfected cells is still biologically active, but not translated in vivo, in agreement with data from the [3H]poly(U)hybridization experiment.
Animals↗
RNA thiolation level (incorporation of methionine-S35) in peripheral lymphocytes of donors and patients with chronic lymphatic leukemia.
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Blood Donors↗
Age-dependent changes of nucleic acid labeling in different rat brain regions.
The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-14C]thymidine incorporation into DNA of cerebral cortex and cerebellum during aging was observed. The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging. In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A) RNA and poly(A) RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.
Aging↗
Clinical correlates of in vitro effect of adriamycin on advanced lung carcinoma.
The effect of 1.38 X 10(-5) M adriamycin on tritiated uridine incorporation was studied after 3 hours treatment of suspensions of 25 advanced human lung carcinomas in vitro. The results were correlated with the responses to clinical therapy. All tumours which showed an inhibition of uridine incorporation in vitro of more than 40% were also sensitive to clinical treatment with adriamycin.
Cells, Cultured↗
[Resistance testing of cytostatic agents on human tumors (author's transl)].
An in vitro short term incubation of human tumors with different cytostatic agents and their corresponding radioactive precursors of cell metabolism allows detection of those drugs which are inefficacious on the examined tumor. The pretherapeutical knowledge of those substances keeps the patient from unnecessary and damaging cytotoxic treatment. The in vitro and in vivo correlation of this technique was tested on 3 different groups of tumor patients: 1. Chemotherapeutically treated tumor patients with primary or secondary induced resistance against the applied cytostatic agents: all substances which clinically did not influence the tumor growth at the moment of the test also were inefficient in the in vitro test system. 2. Tumor patients who were treated according to a clinical therapy regimen contrary to the results of the in vitro testing: corresponding to the test, no influence on tumor growth was seen. 3. Tumor patients who were treated according to the results of the resistance test: after 8 weeks observation none of these patients had any signs of tumor progression.
Antineoplastic Agents↗
Biosynthesis of subviral oncogenic particles (virosomes) in mitochondria of Rous sarcoma and Rauscher murine leukemia cells.
Experimental evidence for the presence and biosynthesis of subviral, leukemogenic particles in the isolated mitochondria of spleen cells of mice infected with Rauscher murine leukemia (RML) virus is presented. These subviral particles sediment at a density of 1.27-1.29 g/ml and induce splenomegaly and RML three weeks after i.v. or i.p. administration to white mice. Virosomes have been labelled with [32P]phosphate in the isolated mitochondria from RML spleen cells and high molecular weight (70S) [32P]RNA has been isolated from these subviral, leukemogenic particles. Rauscher virus group specific antigens were detected by immunodiffusion in the inner membrane and matrix fraction of the mitochondria of RML spleen cells. These results together with our earlier findings strongly suggest that mitochondria of the transformed cells participate in the biosynthesis of RNA tumor viruses. Possible mechanism of the penetration of viral genetic information of RNA tumor viruses into mitochondria of tumor cells in vivo is discussed.
Animals↗
Overexpression of c-Ki-ras and c-fos in human pancreatic carcinomas.
The mRNA expression of protooncogenes c-Ki-ras, c-myc, and c-fos was studied in five pancreatic carcinomas and five normal pancreatic tissues using RNase protection assay and Northern blot analysis. The expression of those protooncogenes was detected in total mRNA from all specimens. However, the amounts in carcinomas and in normal tissues differed. C-Ki-ras mRNA in all the tumors was expressed up to sixfold more than in normal tissues. C-fos mRNA was also overexpressed up to tenfold in four of five tumors. In contrast, c-myc mRNA levels were varied and did not differ significantly between tumors and normal tissues. The results suggest that the overexpression of c-Ki-ras and c-fos mRNA are implicated in the development of pancreatic adenocarcinoma.
Adenocarcinoma↗
Structural alterations of the ribosomal RNA genes in leukemic cells.
Cloned 6.7 kb EcoR1 fragment of mice rDNA was used as a hybridization probe for rDNA structure analysis in mice, rat and calf haemopoietic tumor and normal cells. EcoR1, BglII and Pst1 restriction fragment length polymorphism (RFLP) was found in neoplastic rDNA and was not revealed in normal ones. The rRNA gene rearrangements were observed not only in spacer region but in coding sequences of the genes. Leukemic cells reveal also rDNA amplification. A role of genetic rearrangements of rDNA for mechanisms of carcinogenesis is suggested.
Animals↗
Detection of alpha-methylacyl-coenzyme-A racemase transcripts in blood and urine samples of prostate cancer patients.
BACKGROUND: Alpha-methylacyl-coenzyme-A racemase (AMACR) has been shown to be a highly specific marker for prostate cancer cells, even in the earliest stages of malignant progression. It is expressed at much higher levels than prostate-specific antigen (PSA) in malignant tissues, and is not expressed at appreciable levels in normal prostatic epithelium. In this study, we demonstrate the quantitative detection of AMACR transcripts in peripheral blood of prostate cancer patients using real-time RT-PCR. In addition, we have undertaken a pilot study to demonstrate the potential application of this technique for the detection of prostate tumor cells in urine samples from patients with prostate cancer. METHODS: A real-time RT-PCR assay was developed for detection of the expression of AMACR in prostate cancer patients. Blood samples from 163 patients were tested at various stages of disease progression, with or without therapy. Blood specimens from patients with benign prostate disorders and other types of cancer were also evaluated. RESULTS: In 28 of 58 samples from patients with known metastatic disease who were undergoing treatment, an AMACR expression signal above the cut-off value was detected, consistent with the presence of circulating tumor cells. In 39 of 88 patients with presumptive organ-confined disease, there was evidence of low levels of circulating tumor cells. Comparison of AMACR RT-PCR with known serum PSA values indicated that a combination of these parameters significantly increased the sensitivity for detection of progressive disease. In a pilot study analyzing urine samples from seven prostate cancer patients, elevated AMACR expression levels were detected in the urine sediments of four of six stage-T1 prostate cancer patients and in the one patient with stage-T2 prostate cancer. CONCLUSION: The data presented in this study indicates that AMACR real-time RT-PCR may aid in the detection and staging of prostate cancer.
Adult↗
Detection of a novel truncated WT1 transcript in human neoplasia.
BACKGROUND: The Wilms' tumor 1 (WT1) gene encodes a transcription factor critical in urogenital development. Using a new model of prostate cancer progression that permits comparison of the cellular and molecular properties of increasingly aggressive sublines of simian virus 40 large T-antigen-immortalized human prostate epithelial cells within the same lineage, the role of WT1 in tumorigenesis was investigated. METHODS AND RESULTS: Using RT-PCR and northern blotting, we identified a novel truncated WT1 transcript in these prostate cancer cell lines. This 2.1-kb transcript consisted of the coding region of the zinc-finger domain of WT1, together with a portion of intron 5 at the 5' end of the transcript. Furthermore, two peptides were detected by western blotting using antibodies to epitopes of the COOH terminus of WT1. Using RT-PCR, the 2.1-kb transcript was also detected in leukemia cell line K562, breast cancer cell line MCF7, and blood samples from patients with acute leukemia. CONCLUSION: These novel findings in both cell lines and patient-derived specimens suggest this new WT1 gene alteration has a potential role in the development of new diagnostic assays for some human malignancies.
Blotting, Northern↗
No mutations in TPIT, a corticotroph-specific gene, in human tumoral pituitary ACTH-secreting cells.
BACKGROUND: TPIT is a recently identified transcription factor specific to proopiomelanocortin (POMC)-expressing cells within the pituitary and plays a pivotal role in the embryonal development of POMC lineage. As with other transcription factors, TPIT could theoretically also be involved in corticotroph adenomatous transformation and ACTH hypersecretion and published data indicate that TPIT is present in normal and adenomatous human corticotrophs. OBJECTIVE: The aim of the present study was to corroborate this finding and to seek evidence for mutations in the TPIT coding sequence in human tumoral corticotrophs. DESIGN AND METHODS: Eight human ACTH-secreting pituitary adenomas were collected during surgery, mRNA extracted from primary cultures and reverse transcribed. PCR was performed using 8 different sets of overlapping intron-spanning primers comprising the entire coding sequence of the gene and PCR products analyzed by sequencing. RESULTS: TPIT mRNA was detected in all 8 ACTH-secreting pituitary adenomas without apparent mRNA variants. The entire coding sequence was accounted for, as attested by amplification with all sets of primers. Lastly, sequencing did not reveal differences in the nucleotide arrangement compared with the published sequence. CONCLUSIONS: Aberrant TPIT is unlikely to play a role in corticotroph tumoral trasformation, ie, Cushing's disease, as the entire coding sequence is expressed without any mutation by human pituitary ACTH-secreting adenomas. Conversely, the significance of this transcription factor in tumoral ACTH hypersecretion remains to be clarified.
Adrenocorticotropic Hormone↗
Expression of gonadotropin-releasing hormone (GnRH) and GnRH receptor mRNA in prostate cancer cells and effect of GnRH on the proliferation of prostate cancer cells.
The purpose of this study was to determine the production of gonadotropin-releasing hormone (GnRH), the co-occurrence of GnRH receptors in prostate cancer cells, and the effect of GnRH on prostate cancer cell proliferation. Four human prostate cancer cell lines were studied. LNCaP is an androgen sensitive prostate cancer cell line, DU-145 and PC-3 are androgen resistant, and TSU-Pr1 is uncharacterized. The expression of GnRH and GnRH receptor mRNAs were assessed by in situ hybridization and the effect of exogenous GnRH on proliferation of prostate cancer cells was measured by thymidine incorporation assay. GnRH mRNA expression, determined by in situ hybridization, was found in 83.48% of the LNCaP, 89.7% of the TSU-Pr1, 86.2% of the PC-3 and 95.3% of the DU-145. Signals of GnRH receptor mRNA were detected in more than 95% of the cells of all four cell lines. The proliferation of the prostate cancer cells grown in media supplemented with peptide hormone lacking charcoal-stripped serum was significantly (P < 0.05) suppressed. No significant effect of GnRH on the proliferation of all four prostate cancer cells was observed. In summary, prostate cancer cells produced GnRH and its receptors, and exogenous GnRH treatment did not affect the prostate cancer cell proliferation. The existence of GnRH and GnRH receptor mRNA in the same cell suggests that the role of GnRH produced by prostate cancer cells would be autocrine.
Cell Division↗
Expression of the gene for the multidrug resistance-associated protein in human prostate tissue.
To characterize the clinical relevance of MRP gene in the chemoresistance of prostate carcinomas we determined the multidrug resistance-associated protein (MRP) expression in 30 samples from organ-confined prostate carcinoma, 9 samples from adjacent normal tissue and 4 hormone unresponsive cancers. The measurement of MRP expression was carried out by reverse transcription polymerase chain reaction (RT-PCR) in combination with capillary electrophoresis. Incorporated fluorescence-labeled primers were disclosed by a laser-operated fluorescence detection module. MRP expression was quantified by integration of the peak area and correlated to the ubiquitously expressed beta2 microglobulin. As positive control served the adriamycin-resistant HL60-ADR cell line, which overexpresses MRP. MRP expression was found in all samples. All samples showed a lower MRP/beta2 ratio than HL60-ADR cells. The expression of the MRP gene was 30% higher in organ-confined tumors than in hormone-unresponsive anaplastic tumors. Normal tissue showed the same MRP mRNA level as the adriamycin-sensitive HL60 cells. A higher tumor stage correlated with an increase of MRP expression (> factor 2), whereas G3 tumors displayed a MRP expression 30% lower than in G2 tumors. The small alterations indicate that MRP expression seems not be involved in the chemoresistance of prostate carcinomas.
ATP-Binding Cassette Transporters↗
Cytokines IL-1 alpha, IL-6, and GM-CSF constitutively secreted by oral squamous carcinoma induce down-regulation of CD80 costimulatory molecule expression: restoration by interferon gamma.
We previously characterized the expression of CD80 in different murine head and neck squamous cell carcinoma (HNSCC) clones derived following tumor progression in the absence of T cell-mediated immunity in severe combined immunodeficient (SCID) mice. We found that HNSCCs that did not express CD80 grew as progressors, while those that expressed CD80 were regressors when grown in immune-competent animals. In the present study, we characterized expression of a repertoire of immunoregulatory cytokines in these HNSCC lines, and found that HNSCCs that express cytokines IL-1 alpha, IL-6, and GM-CSF do not express CD80, suggesting the hypothesis that these cytokines may down-modulate expression of CD80. Cytokine-conditioned medium from progressor HNSCC and recombinant IL-1 alpha, IL-6, and GM-CSF caused a reduction of CD80 expression in regressor HNSCCs without affecting proliferation. Conversely, the decrease in CD80 expression in progressor HNSCCs could be restored by IFN-gamma, a known inducer of CD80 expression. These data strongly suggest that high levels of cytokines IL-1 alpha, IL-6, and GM-CSF expressed by tumor cells can down-regulate CD80 expression in HNSCC, and that IFN-gamma can independently stimulate expression. These data provide evidence for a novel mechanism of cytokine-mediated down-modulation of CD80 during malignant progression of HNSCC that can be restored by IFN-gamma.
Animals↗