Growth inhibition by a triple ribozyme targeted to repetitive B2 transcripts.

The B2 family represents a group of short repetitive sequences that are found throughout the rodent genome and are analogous to the human Alu sequences. Certain B2 subfamilies are transcribed by RNA polymerase III (pol III), and this transcription is in part controlled by the retinoblastoma protein. In addition to their putative role in retrotranspositional events, these actively transcribed B2 RNAs show a predicted highly stable secondary structure. Although B2 transcripts are normally confined to the nucleus, they demonstrate altered compartmentation after carcinogen treatment, in cancers, and in immortalized and/or transformed cell lines, the significance of which is unclear. Because modulation of B2 transcripts did not seem feasible with an antisense approach, we designed a triple ribozyme (TRz) construct to down-regulate B2 transcripts. The B2-targeted TRz undergoes efficient self-cleavage, resulting in liberation of the internal hammerhead Rz, which we targeted to a single-stranded region of the consensus B2 sequence. The liberated internal targeted Rz was 20 times more active than the corresponding double-G mutant construct that could not undergo self-cleavage, and 5 times more active than the same Rz flanked by nonspecific vector sequences. The B2-targeted TRz was used to develop stable transfectant clones from an SV40-immortalized hepatocyte cell line. These transfectant clones all showed variably reduced growth rates, accompanied by significant reductions in both cytoplasmic and nuclear B2 RNA levels: linear regression analyses showed that their growth rates were directly related to residual cytoplasmic B2 levels. Reverse-transcription polymerase chain reaction (RT-PCR) analyses documented efficient self-liberation of the internal targeted Rz in vivo, and showed that the relative cytoplasmic expression levels generally paralleled the magnitude of the decrease in B2 transcripts. The RT-PCR analyses further demonstrated that up to 20% of the Rz was located in the nucleus, which presumably reflects competition between autocatalytic processing and nucleocytoplasmic transport of the initial TRz transcript.

Triple ribozyme-mediated down-regulation of the retinoblastoma gene.

We have been developing triple ribozyme (TRz) constructs which consist of two cis-acting ribozymes flanking an internal trans-acting ribozyme, which is targeted to a cellular RNA. Actions of the two cis-acting ribozymes efficiently liberate the internal ribozyme with minimal non-specific flanking sequences. The liberated internal targeted ribozyme shows substantially greater catalytic activity than TRz preparations, constructs which cannot undergo self-liberation or than single ribozymes with flanking vector sequences. Here we construct a TRz which was targeted to retinoblastoma gene (Rb) mRNA, which cleaved Rb target RNA in vitro as expected. A number of tetracycline-regulatable clones stably transfected with the Rb-targeted TRz were developed and analyzed. The internal targeted ribozymes were efficiently liberated in vivo and the stably transfected clones showed varied reductions in Rb mRNA, which were contingent upon ribozyme expression and catalytic activity. The two clones showing major reductions in Rb mRNA (and pRb) levels (>70% reduction) showed abnormal morphology, loss of contact inhibition and the ability to grow in soft agar, as well as altered compartmentation of repetitive B2 transcripts, a phenomenon previously associated with immortalization and/or transformation. TRz constructs coupled with tissue-specific promoters should allow development of in vivo models in which Rb function is markedly reduced in a tissue-specific manner.

Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.

Catecholestrogens are postulated to contribute to carcinogenesis by causing DNA damage mediated by reactive oxygen species generated during redox cycling between catechol and quinone estrogens, and by quinone estrogens that can form depurinating adducts. The above hypothesis is based principally on studies of the cancers that develop in renal cortex of hamsters treated with primary estrogens: Hamster kidney can catalyze 2- and 4-hydroxylation of estrogens and support their redox cycling, and the kidneys of estradiol-treated hamsters show evidence of oxidative cellular and DNA damage. Here we used immunocytochemisty to test the postulate that catechol-O-methyltransferase (COMT), the enzyme that can prevent oxidation of catecholestrogens to their quinone derivatives, would be induced in renal cortex of hamsters treated with estradiol or ethinyl estradiol. In kidneys of control hamsters, COMT was localized in cytoplasm of epithelial cells of proximal convoluted tubules, predominantly in the juxtamedullary region where the estrogen-induced cancers arise. After 2- or 4-weeks of treatment with either estrogen, COMT was seen in epithelial cells of proximal convoluted tubules throughout the cortex, and many cells also showed intense nuclear COMT immunoreactivity. Estradiol-induced renal cancers were negative for COMT, but were surrounded by tubules with intense cytoplasmic and nuclear immunostaining. The nucleus-associated COMT was shown by immunoblot analysis to be the soluble form of the enzyme. Using reverse transcription-polymerase chain reaction amplification, hamster kidney COMT was shown to lack the putative nuclear localization signal sequence present in human COMT. A second phase II enzyme, CuZn-superoxide dismutase (CuZnSOD), was shown by immunocytochemistry to remain extranuclear in proximal convoluted tubules of estrogen-treated hamsters, which indicates entry of COMT into the nucleus to be selective. The findings are consistent with the catechol/quinone estrogen hypothesis of estrogen-induced cancer, while the translocation of the enzyme to the nucleus following estrogen treatment suggests a response to a threat to the genome by electrophilic products of catechols.

Focal nuclear hepatocyte response to oxidative damage following low dose thioacetamide intoxication.

Rats were treated with low doses of the hepatocarcinogen thioacetamide. Forty-eight hours following this treatment, microscopic foci of hepatic injury were observed, which were surrounded by a peripheral rim of histologically normal hepatocytes. These peripheral hepatocytes generally contained enlarged nuclei, and showed nuclear staining for 4-hydroxynonenal-protein adducts, indicative of nuclear oxidative damage. In these same hepatocytes, we also observed specific focal nuclear induction of mu-class glutathione-S-transferase and alcohol dehydrogenase I, two enzymes which are important in metabolism of 4-hydroxynonenal. Of particular interest was the concurrent nuclear induction of APE/ref-1, a multifunctional DNA repair enzyme which can function as a redox factor, and of the transcription factor Jun, whose DNA binding is facilitated by APE/ref-1. These results document an orchestrated focal nuclear response to oxidative damage produced by thioacetamide administration, and may relate to the permanent effects produced by this treatment.

Nuclear multicatalytic proteinase subunit RRC3 is important for growth regulation in hepatocytes.

Multicatalytic proteinases (MCPs) are macromolecular structures involved in the intracellular degradation of many types of proteins. MCPs are composed of a 20S "core" of both structural (alpha) and presumed catalytic (beta) subunits, in association with regulatory proteins. They are characteristically found in both the nucleus and cytoplasm of cells, although mechanisms governing the subcellular distribution of MCPs are not known. RRC3, an alpha subunit of rat MCPs, contains both a putative nuclear localization signal (NLS) and a potential tyrosine phosphorylation site which could play a role in nuclear import, and the nuclear form of RRC3 appears to be involved in the regulation of cell growth. Here we have generated a variety of RRC3 expression constructs to study features of RRC3 important in nuclear localization and cell growth. PCR was utilized to develop constructs containing point mutations in either the putative NLS (K51 mutated to A) or at a potential tyrosine phosphorylation site (Y121 mutated to F), and an epitope from influenza hemagglutinin (HA) was added in triplicate to the C-terminus of the constructs as a means of identification. RRC3 constructs were then made in which the nucleotide sequence near the translation initiation site of RRC3 was modified in such a way that the amino acid sequence of the protein translated from the constructs is unchanged from that of normal RRC3, thus allowing differential modulation of endogenous RRC3 with antisense oligonucleotide treatment. These N-terminally modified constructs are designated mC3, mC3NLS, and mC3y. In vitro transcription/translation reactions with these constructs produced the expected products, which were immunoprecipitated with a mouse monoclonal anti-HA antibody. Immunohistochemical studies with hepatocyte cell lines transiently transfected with either mC3NLS or mC3y showed only cytoplasmic staining, whereas cells transfected with mC3 had a staining pattern typical of endogenous RRC3 (both cytoplasmic and nuclear) with strong staining of the nuclear perimeter. Immunoblot analyses of subcellular fractions from stably transfected CWSV1 cells showed mC3 product in both the cytosol and nucleus of cells, whereas mC3NLS or mC3y products were restricted to the cytosol. CWSV1 cells stably transfected with the pTet-Splice vector containing no insert (as a control) were markedly inhibited (80%) in cell growth and showed altered morphology when treated with antisense oligonucleotides targeted to endogenous RRC3, reproducing previous studies. Similarly, CWSV1 cells stably transfected with either mC3NLS or mC3y constructs showed analogous growth inhibition and morphologic alteration upon antisense treatment. In contrast, CWSV1 cells stably transfected with the mC3 construct showed normal growth and morphology following antisense oligonucleotide treatment, demonstrating that replenishment of nuclear RRC3 was necessary and sufficient to relieve growth inhibition. In 32P-metabolic labeling studies, mC3 was tyrosine-phosphorylated in cytosol as the full-length protein (M(r) 36000). mC3NLS was also phosphorylated in cytosol, whereas mC3y was not. Nuclear mC3 showed phosphorylation of a M(r) 27000 processed form while neither mC3NLS nor mC3y showed any phosphorylated nuclear products. Our results show that nuclear RRC3 is important in control of cell growth and that both the NLS and Y121 are important in nuclear localization of RRC3. Control of nuclear import by tyrosine phosphorylation may represent a novel regulatory mechanism, and our results further suggest that RRC3 may travel as a maverick subunit.

Nuclear multicatalytic proteinase alpha subunit RRC3: differential size, tyrosine phosphorylation, and susceptibility to antisense oligonucleotide treatment.

Multicatalytic proteinases (MCPs) are macromolecular structures involved in intracellular degradation of many types of proteins. MCPs are composed of a 20S "core" which consists of both structural (alpha) and presumed catalytic (beta) subunits in association with complexes of accessory proteins. Immunohistochemical studies have shown MCP subunits to be largely cytoplasmic, although nuclear localization is also observed. Reverse transcription/polymerase chain reaction amplifications were performed with redundant primers to conserved regions within known subunits, in an attempt both to identify potential new subunits and to define the repertoire of subunits expressed in hepatocytes. No new subunits were identified, and we found that RRC3, an alpha subunit of MCPs which contains a putative nuclear localization signal (NLS), was the predominant alpha subunit expressed in hepatocytes and hepatocyte-derived cell lines. Antibodies were developed against a unique C-terminal peptide region of RRC3. Immunohistochemical studies using affinity-purified antibodies showed that RRC3 has both cytoplasmic and nuclear localizations. Immunoprecipitation/immunoblot analyses showed that a significant proportion of nuclear RRC3 was associated with the nuclear scaffold (NS). NS RRC3 showed a significantly smaller M(r) (24,000) than the cytoplasmic form (M(r) 28,000), and only the nuclear form contained phosphotyrosine. In metabolic labeling experiments with [32P]orthophosphate, the major nuclear and NS form observed showed an M(r) of 24,000, whereas no labeling of cytosolic RRC3 was observed. A minor 32P-labeled band of M(r) 28,000 was also observed in nuclei, and this M(r) 28,000 form was found in the soluble nuclear extract within MCP complexes. These results suggest that tyrosine phosphorylation of the cytosolic form (M(r) 28,000) rapidly triggers nuclear import, which is in turn quickly followed by conversion to the major M(r) 24,000 form associated with NS. Treatment with antisense oligonucleotides targeted to the initiation site of RRC3 reduced the growth of a hepatocyte-derived cell line by 95% and produced a marked morphological change (in the absence of overt toxicity). Under these treatment conditions, RRC3 mRNA was dramatically reduced. RRC3 protein was also dramatically reduced in the NS, but showed only a small reduction in cytosol, suggesting that the nuclear RRC3 may be important in cell growth and differentiation.

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species.

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis-regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.