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At least 1,063 records · Page 59Linked to original sources
PubMed1995

Suppression of tumorigenesis by transcription units expressing the antisense E6 and E7 messenger RNA (mRNA) for the transforming proteins of the human papilloma virus and the sense mRNA for the retinoblastoma gene in cervical carcinoma cells.

Human cervical carcinoma cell lines that harbor human papilloma virus (HPV) have been reported to express HPV E6 and E7 proteins at least in the beginning stages if not at all stages of the disease. The HPV E6 and E7 proteins bind to and inactivate the products of the p53 and retinoblastoma (Rb) tumor suppressor genes, which thereby allow the cervical carcinoma cells to circumvent the action of these tumor suppressor genes. We observed that the introduction of the antisense HPV 18 E6 and E7 sequences, as well as a sense cDNA for the human wild-type Rb gene into a human cervical carcinoma cell line (HeLa), which is positive for the HPV 18 provirus, decreased the in vitro and in vivo growth rate of the transfected cells if both antisense transcripts for the HPV 18 E6 and E7 and sense transcripts for human Rb were expressed. In addition, overexpression of a complementary DNA (cDNA) for the Rb messenger RNA was sufficient to slow the proliferation of HeLa cells, and the level of Rb cDNA expression was correlated with the degree to which the rate of growth of the tumor was slowed. The results of our experiments show that the presence of HPV E6 and E7 proteins and the resultant inactivation of Rb in cervical carcinoma cells contributes to the neoplastic phenotype even in highly evolved cervical carcinoma cell lines such as HeLa, which have been derived from a cervical carcinoma patient at an advanced stage of the disease process. These data suggest that the HPV proteins play a role not only at the beginning of cervical cancer, but also at advanced stages of this disease. These experiments may lead to genetic approaches to the control of this disease that involve antisense sequences that downregulate the E6 and E7 genes or lead to expression of the Rb gene.

Animals↗
PubMed1994

Trk mRNA and low affinity nerve growth factor receptor mRNA expression and triploid DNA content in favorable neuroblastoma tumors.

Nerve growth factor (NGF) is suggested to play a role in spontaneous differentiation or regression of neuroblastoma. Expression of mRNAs for trk protooncogene and NGF low affinity receptor gene (LNGFR) were analyzed in 45 neuroblastomas with Northern Blot. Trk and LNGFR expression correlated with young age, localized tumors or IV-S disease, absence of N-myc amplification, triploid DNA content, spontaneous tumor regression and favorable prognosis. Regardless of other factors, trk and LNGFR mRNAs distinguished three prognostic subsets: one favorable (trk+, LNGFR+, n = 19, 100% survival probability), one intermediate (trk+, LNGFR-, n = 11, 62%) and one poor (trk-, n = 15, 0%). An algorithm based on the combined analysis of trk and LNGFR mRNAs and DNA ploidy divided the material in two groups with 96 and 0% survival probability respectively (p < 0.001), and predicted outcome accurately in 43 of 45 children. It is hypothesized that loss of functional NGF-receptors constitutes an early critical step in the evolution of unfavorable neuroblastomas prone to progression in spite of current therapy.

DNA, Neoplasm↗
PubMed1997

Estrogen receptor-beta mRNA expression in rat ovary: down-regulation by gonadotropins.

We have examined the expression and regulation of the two estrogen receptor (ER alpha and ER beta) genes in the rat ovary, using Northern blotting, RT-PCR, and in situ hybridization histochemistry. Northern blotting results show that the ovary expresses both ER alpha and ER beta genes as single (approximately 6.5-kb) and multiple (ranging from approximately 1.0-kb to approximately 10.0-kb) transcripts, respectively. ER alpha mRNA is expressed at a level lower than ER beta mRNA in immature rat ovaries. This relationship appears unchanged between sexually mature adult rats and immature rats. In sexually mature adult rats undergoing endogenous hormonal changes, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less constant with the exception of the evening of proestrus when ER beta mRNA levels were decreased. Examination of ER beta mRNA expression at the cellular level, by in situ hybridization, showed that ER beta mRNA is expressed preferentially in granulosa cells of small, growing, and preovulatory follicles, although weak expression of ER beta mRNA was observed in a subset of corpora lutea, and that the decrease in ER beta mRNA during proestrous evening is attributable, at least in part, to down-regulation of ER beta mRNA in the preovulatory follicles. This type of expression and regulation was not typical for ER alpha mRNA in the ovary. Although whole ovarian content of ER alpha mRNA was clearly detected by RT-PCR, no apparent modulation of ER alpha mRNA levels was observed during the estrous cycle. Examination of ER alpha mRNA expression at the cellular level, by in situ hybridization, showed that ER alpha mRNA is expressed at a low level throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ER beta mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ER beta mRNA. In situ hybridization for ER beta mRNA shows that small, growing, and preovulatory follicles express ER beta mRNA in the granulosa cells. The preovulatory follicles contain ER beta mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ER beta mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ER beta mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ER beta mRNA in cultured granulosa cells. Taken together, our results demonstrate that 1) the ovary expresses both ER alpha and ER beta genes, although ER beta is the predominant form of estrogen receptor in the ovary, 2) ER beta mRNA is localized predominantly to the granulosa cells of small, growing, and preovulatory follicles, and 3) the preovulatory LH surge down-regulates ER beta mRNA. These results clearly implicate the physiological importance of ER beta in female reproductive functions.

Age Factors↗
PubMed1987

Attachment of mRNA to the cytoskeletal framework and translational control of gene expression in rat L6 muscle cells.

The mRNA of rat L6 muscle cells was distributed between a detergent-insoluble fraction containing the cytoskeletal framework and a detergent-soluble fraction. The majority of cytoskeleton-bound mRNA was translationally active and present as polysomes. The mRNA of the detergent-soluble fraction was not associated with the ribosomes and, thus, considered to be the repressed free population. The binding of mRNA was not mediated through ribosomes or the poly(A) region of mRNA. Cross-linking of RNA and proteins was used to examine whether proteins of the cytoskeletal framework were involved in binding mRNA to this structure. Analysis of the mRNA-protein complexes has shown that a large number of polypeptides of molecular masses between 15 and 220 kilodaltons (kDa) were associated with both cytoskeleton-bound and soluble mRNAs. However, a 165-kDa polypeptide was preferentially associated with cytoskeleton-bound mRNA-protein complexes. This polypeptide was also enriched in the total proteins of the cytoskeleton fraction. We have suggested a receptor-like role for the 165-kDa polypeptide in binding mRNA to the cytoskeletal framework. The mechanism of interaction between the cytoskeleton and mRNA was further examined by using a ghost-monolayer transcription system. The mRNA synthesized by this transcription system was preferentially retained in the detergent-insoluble cytoskeleton component of the ghost-monolayer preparation. To understand the physiological significance of the distribution of mRNA between the translationally active cytoskeleton-bound and repressed soluble fractions we have isolated a cDNA clone for a 1.3-kilobase (kb) mRNA. This mRNA was preferentially repressed in myotubes. Distribution of this mRNA was determined by Northern blot analysis using the recombinant plasmid. This analysis indicates that nearly 90% of this mRNA was not associated with ribosomes. In contrast, only 3% of alpha-actin mRNA was found in the repressed population. However, approximately 25% of the 1.3-kb mRNA was present as repressed free messenger ribonucleoprotein. This behaviour is again different from that of actin. All of the cytoskeleton-bound alpha-actin mRNA was associated with polysomes. Furthermore, most of the small amount of alpha-actin mRNA which was present in the soluble fraction was also associated with polysomes. We have, therefore, concluded from these observations that binding of mRNA to the cytoskeleton framework and translation of mRNA are two separate events. We have suggested that mRNA is transported to the cytoplasm as a cytoskeleton-associated complex and further interaction with ribosomes may stabilize this complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Regulation of sulfotransferase mRNA expression in male and female rats of various ages.

Sulfotransferases (SULTs) are Phase II drug-metabolizing enzymes that catalyze the addition of a sulfuryl moiety to both endogenous compounds, including steroids and neurotransmitters, and certain xenobiotics, including N-hydroxy-2-acetylaminoflourine and phenolic compounds, like alpha-naphthol. In contrast to certain Phase I drug-metabolizing enzymes, little is known about the regulation of the sulfotransferases. These series of studies were designed to analyze SULT mRNA expression and hormonal regulation in male and female rats. The hepatic expression of six different SULT isoforms was examined including three phenol SULTs and three hydroxysteroid SULTs. SULT mRNA expression was examined in adult and developing rats, as well as, in hypophysectomized (HX) and growth hormone-supplemented HX animals. SULT1A1 is thought to be important for the sulfation of simple phenols and its mRNA expression is about twice as high in adult male as in female rats. This difference in SULT1A1 mRNA levels is largely due to a greater decrease in mRNA levels after puberty in female than in male rats. Hypophysectomy resulted in a decrease in expression of SULT1A1 mRNA in both male and female rats. Replacement of growth hormone (GH) by either intermittent injection (male pattern) or infusion (female pattern) failed to restore SULT1A1 expression. Sulfotransferase SULT1C1 has been implicated in activation of N-hydroxyacetylaminoflourine. In contrast to SULT1A1, SULT1C1 mRNA expression is about 10-fold higher in adult males than in adult female rats. This male-dominant expression pattern emerges at 40-50 days of age and is due to an increase in SULT1C1 mRNA in males. Hypophysectomy abolished SULT1C1 expression in male rats. Interestingly, replacement of GH by injection (male pattern) restored SULT1C1 mRNA expression in males and enhanced SULT1C1 expression in female rats to levels observed in adult male rats. GH infusion (female pattern) did not affect SULT1C1 mRNA expression in either male or female rats. Estrogen sulfotransferase (SULT1E2) may play a role in estrogen homeostasis. Adult male rats express SULTIE2 mRNA at levels 10-fold higher than those observed in adult females and similar to SULT1C1, this is due to an increase in SULT1E2 mRNA occurring during puberty in the male rat. Hypophysectomy did not appreciably affect SULT1E2 expression in male rats, however in contrast to males, hypophysectomy markedly enhanced SULT1E2 expression in female rats. GH infusion suppressed SULT1E2 levels in HX male rats. The expression of hydroxysteroid sulfotransferases was also examined. The SULT-20/21 isoform was expressed in both male and female rats. Male expression of this isoform peaked at 30 days of age and then declined to approximately 30% of the level observed in adult females. SULT-20/21 mRNA expression increased sharply at 45 days of age in female rats and remained elevated. Expression of SULT-20/21 mRNA was decreased markedly by hypophysectomy in both male and female rats. GH injection did not affect SULT-20/21 mRNA expression in HX males, however this treatment resulted in a 4-fold increase in SULT-20/21 mRNA in HX females. GH infusion restored SULT-20/21 expression in HX-male rats. GH infusion did elevate SULT-20/21 mRNA expression in female-HX rats, but not to the level observed in intact females. Hydroxysteroid SULT isoform SULT-40/41 was expressed in adult female but not adult male rats. SULT-40/41 expression peaked at 15 days of age in both male and female rats and decreased thereafter. The decrease in expression was more pronounced in male rats. SULT-60 mRNA, like SULT-40/41, was expressed only in adult female rats. Male rats express SULT-60 at 30 days of age, but SULT-60 mRNA is undetectable at 60 days. SULT-60 mRNA was expressed in females only after day 30 and female SULT-60 mRNA expression remains high thereafter. SULT-40/41 and SULT-60 mRNA expression was increased by HX in male rats and decreased by HX in female rats. (ABSTRACT TRUNCATED)

Aging↗
PubMed1996

Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome.

The codon UGA located 5' adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli. The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNA(Sec) and GTP. We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5' adjacent to the fdhF mRNA hairpin. By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time. Toeprint analysis of the mRNA-ribosome complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site. Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track. Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNA(Sec) was used instead of tRNA(fMet). Lack of binding of selenocysteinyl-tRNA(Sec) to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin. By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivo when fused in-frame 5' of the lacZ gene. Therefore, our data indicate that the fdhF mRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNA(Sec)-GTP complex.

Aldehydes↗