Inhibition of papilloma progression by antisense oligonucleotides targeted to HPV11 E6/E7 RNA.

Human papillomaviruses (HPVs) are recognized as important human pathogens, causing a spectrum of hyperproliferative lesions from benign warts to cervical dysplasias/carcinomas. HPV-associated lesions require continued production of the oncogenic E6/E7 proteins, which are encoded by either bicistronic or overlapping mRNAs. Here we targeted the E6/E7 mRNA of HPV11, a type implicated in causation of genital warts, using molecular reagents. Accessible sites in the HPV11(E6/E7) RNA were identified using library selection protocols, and nucleic acids (DNAzymes, antisense oligonucleotides) targeted to these sites were constructed, and tested in cell culture and on human foreskin grafts. While DNAzymes were at least equally effective in cell culture, antisense oligonucleotides targeted to the region surrounding one of the library-selected sites (ASO(407)) proved most effective in blocking progression of HPV11-induced papillomas in human foreskin grafts on immunodeficient mice. In total, 11 papillomas were treated with ASO(407). Of these, four of seven small papillomas treated with ASO(407) showed loss of detectable virus by in situ hybridization (ISH), and in all four of these, papillomas were no longer evident grossly or histologically after treatment. When larger papillomas were treated, one of four showed loss of virus by ISH, associated with a minor decrease in papilloma size. Considering all 11 papillomas treated with ASO(407), loss of viral staining by ISH was significantly different from that observed in controls (P<0.016), as was true for the seven small treated papillomas (P<0.012). DNAzymes targeted to the same site (or other library selected sites) did not produce statistically significant differences in ISH staining (P<0.15). Our results with ASO(407) appear to represent the first specific molecular therapy against a bona fide HPV infection, and provide a rational proof-of-principle strategy for development of molecular therapeutics targeting other HPV-associated lesions.

A selection system for identifying accessible sites in target RNAs.

Although ribozymes offer tremendous potential for posttranscriptionally controlling expression of targeted genes, their utility is often limited by the accessibility of the targeted regions within the RNA transcripts. Here we describe a method that identifies RNA regions that are accessible to oligonucleotides. Based on this selection protocol, we show that construction of hammerhead ribozymes targeted to the identified regions results in catalytic activities that are consistently and substantially greater than those of ribozymes designed on the basis of computer modeling. Identification of accessible sites should also be widely applicable to design of antisense oligonucleotides and DNAzymes.

Genomic instability-based transgenic models of prostate cancer.

To develop animal models that represent the broad spectrum of human prostate cancer, we created transgenic mice with targeted prostate-specific expression of two genes (ECO:RI and c-fos) implicated in the induction of genomic instability. Expression of the transgenes was restricted to prostate epithelial cells by coupling them to the tissue-specific, hormonally regulated probasin promoter (PB). The effects of transgene expression were examined histologically in prostate sections at time points taken from 4 to 24 months of age. The progressive presence of regions of mild-to-severe hyperplasia, low- and high-grade prostatic intra-epithelial neoplasia, and well-differentiated adenocarcinoma was observed in both PBECO:RI lines but no significant pathology was detected in the PBfos line. Prostate tissue of PBECO:RI mice was examined for expression of p53, proliferating cell nuclear antigen (PCNA) and Ki67 at multiple time points. Although p53 does not appear to be mutated, levels of PCNA and Ki67 are elevated and correlate with the severity of the prostatic lesions. Overall, pre-neoplastic and neoplastic stages represented in the PBECO:RI model showed similarity to corresponding early stages of the human disease. This genomic instability-based model will be used to study the mechanisms involved in the early stages of prostate carcinogenesis and to investigate the nature of subsequent events necessary for the progression to advanced disease.

Nuclear localization of catechol-O-methyltransferase in neoplastic and nonneoplastic mammary epithelial cells.

Catechol-O-methyltransferase (COMT) plays both a regulatory and protective role in catechol homeostasis. It contributes to the regulation of tissue levels of catecholamines and catecholestrogens (CEs) and, by blocking oxidative metabolism of catechols, prevents endogenous and exogenous catechols from becoming a source of potentially mutagenic electrophiles. Evidence implicating CEs in carcinogenesis, in particular in the hamster kidney model of estrogen-induced cancer, has focused attention on the protective role of COMT in estrogen target tissues. We have previously reported that treating hamsters with estrogens causes translocation of COMT to nuclei of epithelial cells in the renal cortex, the site of CE biosynthesis and where the cancers arise. This finding suggested that nuclear COMT may be a marker of a threat to the genome by catechols, including CEs. It is postulated that CEs play a role in the genesis of breast cancer by contributing to a state of chronic oxidative stress that is presumed to underlie the high incidence of this disease in the United States. Therefore, here we used immunocytochemistry to re-examine human breast parenchyma for nuclear COMT. In addition to confirming previous reports of cytoplasmic COMT in mammary epithelial cells, we identified nuclear COMT in foci of mammary epithelial cells in histologically normal breast tissue of virtually all control (macromastia) and cancer patients and in breast cancer cells. There was no correlation between tissue histology and the numbers of cells with nuclear COMT, the size of foci containing such cells, or intensity of nuclear COMT immunostaining. The focal nature of the phenomenon suggests that nuclear COMT does not serve a housekeeping function but that it reflects a protective response to an increased local catechol load, presumably of CEs and, as such, that it may be a characteristic of the population of women studied who share the same major risk factor for developing breast cancer, that of living in the industrialized West.

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.

Expression of SCL is normal in transfusion-dependent Diamond-Blackfan anemia but other bHLH proteins are deficient.

Basic helix-loop-helix proteins, which are tissue specific (SCL) or broadly expressed (E proteins), interact positively to regulate erythroid specific genes. Here, expression of SCL and two broadly expressed E proteins, E47 and HEB, was high early in erythroid differentiation and declined during maturation. Stimulation of erythroid progenitors/precursors with stem cell factor (SCF) enhanced SCL and E protein levels, one mechanism by which SCF may increase erythroid proliferation. Interactions between SCL and E proteins are competed by Id2, which binds and sequesters E proteins. Upregulation of Id2, demonstrated here late in erythroid differentiation, may downregulate genes involved in erythroid proliferation/differentiation. We examined expression of bHLH proteins in transfusion-dependent patients with Diamond-Blackfan anemia (DBA) to determine if these interactions are disrupted. In erythroblasts from patients, expression of SCL protein and mRNA was normal and SCL increased in response to SCF. However, E47 and HEB protein levels were significantly decreased. Id2 was strongly expressed in patients. Through reduction of SCL/E protein heterodimer formation, abnormal levels of bHLH transcription factors may affect expression of erythroid specific genes, such as beta globin. Stimulation of Diamond-Blackfan cells with SCF partially compensated for this defect, enhancing expression of E47, HEB, and SCL. SCF may function to increase SCL/E protein heterodimer formation, which may be one of the mechanisms through which SCF stimulates erythroid proliferation/differentiation in DBA.

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.

Studies on rLMP7, a beta-subunit of the multicatalytic proteinase.

Subunit rLMP7 of the multicatalytic proteinase (MCP), which has been associated with chymotrypsin-like proteinase activity, was examined in rat liver and hepatocyte-derived cell lines. rLMP7 was detected in both nucleus and cytosol in liver by immunohistochemistry and immunoblotting, using a peptide-specific anti-rLMP7 antibody. A M(r) 30,000 precursor protein was present only in cytosol, as was a minor component of M(r) 25,000. Mature rLMP7 (M(r) 23,000) was present in MCP in both nucleus and cytosol, although it was not detectable in the nuclear scaffold. Two rLMP7 cDNAs (designated rLMP7.1 and rLMP7.s) were identified by rapid amplification of 5' ends using RT/PCR, a result which was confirmed by Northern blot analysis and RNase protection assays. rLMP7.1 is 3-4x more abundant than rLMP7.s; it is 50 nt longer than the previously reported cDNA sequence and includes an upstream in-frame ATG within a consensus translation initiation sequence, which encodes the M(r) 30,000 rLMP7 precursor protein identified in vivo. rLMP7.s is 100 nt shorter than rLMP7.1 and does not contain the most 5' ATG. Transient transfection analyses with rLMP7.1 and rLMP7.s constructs coupled to green fluorescent protein showed that both transcripts were efficiently expressed in vivo. In vitro expression of these two rLMP7 cDNAs showed that rLMP7.1 produces the M(r) 30,000 precursor protein, whereas rLMP7.s produces two smaller peptides of M(r) 25,000 and 23,000. Purified 20S MCP preparations were able to proteolytically process the M(r) 30,000 precursor to the M(r) 25,000 product but not to the mature rLMP7 form. However, incorporation of this processed M(r) 25,000 product (or of either M(r) form produced from rLMP7.s) did not occur in vitro. In vitro processing and pulse-chase experiments suggested that the mature M(r) 23,000 subunit is derived, at least in part, from the M(r) 30,000 precursor. The M(r) 25,000 form may be a stable product produced directly from rLMP7.s.

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.

Focal altered compartmentation of repetitive B2 (Alu-like) sequences in rat liver following hepatocarcinogen exposure.

Rats were treated with low doses of the hepatocarcinogens dimethylnitrosamine or thioacetamide, and livers were examined 48 h later. These treatments are known to produce altered RNA compartmentation, wherein a class of repetitive RNA sequences normally restricted to the nucleus appears in the cytoplasm. Reverse transcription-PCR amplifications demonstrated that the sequences showing altered compartmentation consisted largely of a subfamily of the rodent B2 sequence family, the counterpart of human Alu sequences involved in retrotransposition. Northern blot analyses showed that these B2 sequences were found in cytoplasmic RNA as 170- to 360-nucleotide "sense" transcripts, and competition hybridization experiments established that B2 sequences represented most (if not all) of the sequences showing altered compartmentation. The major increase in B2 transcriptions in cytoplasmic RNA was not associated with any change in B2 transcription by RNA polymerase III. In situ hybridizations showed that the altered compartmentation of B2 sequences occurred in well-delineated foci within the rat liver; these foci consisted of a central region containing a prominent infiltrate of macrophages admixed with small hepatocytes and a peripheral region of histologically normal hepatocytes that showed evidence of oxidative damage. Altered compartmentation of B2 sequences may represent an important focal initiatory change in a subset of hepatocytes, whereas subsequent retrotranspositional events (associated with Alu-like sequences) could predispose initiated cell foci to alterations in promotion/progression phases.

Protease inhibitors and carcinogenesis: a review.

This brief review article deals with the subject of anticarcinogenic activity of protease inhibitors (PI). Three basic premises are made: (1) Although PI are prevalent constituents of dietary staples such as soy products, which have been epidemiologically associated with reduced cancer incidences at multiple target sites, they are unlikely to be the active anticarcinogenic entities. Cooked soy products, which are devoid of PI activity, are equally as effective at reducing cancer development as raw soy products. Isoflavones are likely to represent major chemopreventive agents in soy, although other constituents may well contribute. (2) Although supplementation of diets with PI (natural or synthetic), or direct topical administration, results in lower cancer incidences in many experimental models in vivo, this effect appears to be indirect. Dietary PI are, in general, poorly absorbed from the GI tract, and never reach target organs in any measurable quantity. The most attractive hypothesis is that dietary PI could induce synthesis and distribution of endogenous PI (acute-phase reactants), which have widespread effects on cell growth and behavior. Effects of topical administration of PI also encompass prominent anti-inflammatory effects. (3) A spectrum of PI inhibit in vitro transformation induced by a variety of carcinogenic agents. Their effects can be grouped into three basic categories, affecting: (a) signal transduction pathways; (b) DNA repair processes; and (c) nuclear proteases. I suggest that the nuclear multicatalytic protease activity, in particular the chymotrypsin-like activity, represents an important cellular target for which considerable anecdotal support can be garnered.

Nuclear scaffold-associated protease: in situ nuclear localization and effects of a protease inhibitor on growth and morphology of a ras-transformed hepatocyte cell line.

We have previously identified a multicatalytic protease (MCP) activity associated with the nuclear scaffold (NS) in hepatocytes and fibroblasts. When we used the chloromethylketone protease inhibitor AAPFcmk, which is targeted to chymotrypsinlike protease activity, we observed a dramatic inhibition of transformation of fibroblasts, with effects that were relatively selective for the NS fraction. Here, we undertook experiments to determine the effects of AAPFcmk on Simian Virus 40-immortalized CWSV1 cells compared with a ras-transformed hepatocyte cell line (NR4) derived from CWSV1. We used biotinAAPFcmk and fluorescent reagents to demonstrate a nuclear chymotrypsinlike protease activity, which is most prominent at the nuclear envelope. The ras-transformed NR4 cells were highly susceptible to growth inhibition in a dose-dependent manner, showing 85% growth inhibition at 50 mumol/LAAPFcmk. In contrast, the immortalized CWSV1 cells were not sensitive at the concentrations (10 to 50 mumol/L) of AAPFcmk tested. In subcellular fractionation studies, the inhibitory effects of AAPFcmk were confined to the NS fraction. The AAPFcmk-induced growth inhibition was accompanied by marked morphological changes in ras-transformed cells, without evidence of overt toxicity. No change in DNA content was observed, but a marked increase in organization of actin cytoskeletal elements was seen. These results suggest that a protease activity associated with the nuclear scaffold has important functions in controlling cytoskeletal filament organization and cell replication.

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.

An inhibitor of nuclear scaffold protease blocks chemical transformation of fibroblasts.

A nuclear scaffold (NS) protease has previously been implicated in production of the M(r) 46,000 ATP-binding protein in NS (which may acquire nucleoside triphosphatase activity and participate in nucleocytoplasmic transport) by cleavage of a subset of lamins A/C. In a preceding paper (G. Clawson, L. Norbeck, C. Hatem, C. Rhodes, P. Amiri, J. McKerrow, S. Patierno, and G. Fiskum, Cell Growth & Differ., 3: 827-838), this NS protease was identified as a novel, Ca(2+)-regulated serine protease, which was found only in the NS and which appears to represent a unique multicatalytic protease complex. Based upon its predominantly chymotrypsin-like substrate preference, a peptide-chloromethylketone inhibitor (succinyl-AAPF-chloromethylketone, AAPFcmk) was identified. AAPFcmk showed a KI = 56 nM for the NS protease versus 1.4 microM for the endoplasmic reticulum activity. Treatment of C3H/10T1/2 mouse embryo fibroblast cells with 1 microM AAPFcmk produced effects which were confined to the nuclear (and to a lesser extent the endoplasmic reticulum) compartment. In this report, we examine the effects of the AAPFcmk inhibitor on cellular transformation and growth. Growth of C3H/10T1/2 cells was decreased by 34% and 56% at 25 microM and 50 microM AAPFcmk, respectively. Growth inhibition occurred without any major change in DNA content distribution, suggesting effects throughout the cell cycle. Growth inhibition was not observed at lower (< or = 10 microM) concentrations, which decreased transformation of C3H/10T1/2 fibroblasts in a dose-dependent manner by up to 90%, even at femtomolar concentrations of AAPFcmk (in the absence of growth inhibition). Inclusion of irrelevant inhibitors was without affect.(ABSTRACT TRUNCATED AT 250 WORDS)

The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins.

The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.

Ca(2+)-regulated serine protease associated with the nuclear scaffold.

The nuclear scaffold (NS) is a proteinaceous network of orthogonally arrayed intermediate filament proteins, termed lamins, which is responsible for nuclear structure. Recent work has demonstrated that a subset of lamins A/C is proteolytically cleaved to produce an ATP-binding protein. This proteolytic cleavage is accomplished by a NS protease activity, which shows a considerable selectivity for lamins A/C and is stringently regulated by Ca2+ in vitro, suggesting that it might also participate in control of NS breakdown in various scenarios. Here, we identify the major NS protease as a novel serine protease with a predominantly chymotryptic-like substrate preference, and we show that even transient perturbations in cytosolic Ca2+ have significant effects on the NS protease activity. This NS protease activity shows extensive similarities to the multicatalytic proteinase complex. In addition to a potential role in control of NS breakdown at mitosis and/or under pathological conditions, this NS protease is also strategically located for other functions, such as inactivation of various oncogenic proteins or maturation-promoting factor.

Nuclear enlargement induced by hepatocarcinogens alters ploidy.

Treatment of rats with low doses of hepatocarcinogens is associated with a number of phenomena, including nuclear enlargement and altered nucleocytoplasmic compartmentation, which potentially reflect initiatory changes. Since a number of investigations have indicated that changes in ploidy may relate to the development of altered foci and/or hepatocellular carcinomas in liver, and since nuclear enlargement may be associated with changes in DNA content, we examined the ploidy of rat hepatocytes following essentially nontoxic low-dose carcinogen treatment. Treatments with thioacetamide, 2-acetylaminofluorene, or dimethylnitrosamine were all associated with a notable shift of nuclei from diploid to tetraploid, in the apparent absence of nuclear replication. These changes were similar in magnitude to that associated with the substantial liver regeneration induced by carbon tetrachloride. If it is argued that cell replication is a necessary prerequisite for the completion of initiation, these data suggest that there may be thresholds for initiation.