Combining gene and immunotherapy for prostate cancer.

The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in pre-clinical studies and is currently being assessed in phase I and II clinical trials in prostate cancer. Enhanced cell killing by apparent immune-mediated mechanisms has been shown in pancreatic and colorectal cancer models, by co-expressing murine granulocyte macrophage colony-stimulating factor (GM-CSF) with NR in a single replication deficient adenoviral vector. This consists of the CMV immediate early promotor driving expression of NR, with an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF). To examine if similar enhancement of tumour cell killing could be produced in prostate cancer, the TRAMP model was chosen. Results illustrate that the combination of suicide gene therapy using NR and CB1954, with cytokine stimulation with mGM-CSF gives an improved response compared with either modality alone. The mechanism of this improved response is however likely to be non-immune based as it lacks a memory effect.

Incorporation of a laminin-derived peptide (SIKVAV) on polymer-modified adenovirus permits tumor-specific targeting via alpha6-integrins.

Effective gene therapy for disseminated metastatic cancer is currently impossible because of poor delivery of vector to target sites. Modification of viral vectors to target advanced cancer has long been a challenge. In this study, we aimed to redirect adenovirus tropism to infect prostate cancer cells via alpha6beta1 integrins, whose expression is upregulated during prostate cancer progression. To ablate normal mechanisms of infection and provide a framework for attachment of targeting ligands, viruses were non-genetically modified with pHPMA-ONp polymer. Addition of polymer-coated virus to prostate cells showed significantly reduced transgene expression compared with unmodified virus. To restore infectivity, an alpha6-integrin binding peptide (-SIKVAV-) derived from laminin was incorporated onto the surface of the polymer-coated viruses. Photon correlation spectroscopic analysis revealed a small increase in the mean diameter of the particles following retargeting. Addition of -SIKVAV- peptide restored virus infectivity of PC-3 cells in a ligand concentration-dependent manner that was significantly improved following removal of unincorporated polymer and peptide. Competition assays using cells preincubated with Ad5 fiber protein or free -SIKVAV- peptide confirmed that entry of retargeted viruses was mediated via the incorporated ligand. Application of retargeted viruses to a panel of human cell lines revealed varying levels of transduction efficiency. Flow cytometric analysis of cells using anti-alpha6 integrin and anti-beta1 integrin antibodies demonstrated that for prostate cells, greater transduction efficiency correlated with higher levels of expression of both integrin subunits. Furthermore with the exception of LNCaP cells, increased alpha6beta1 integrin expression correlated with advanced disease. Intravenous administration of retargeted viruses to tumor-bearing mice resulted in slower plasma clearance and greatly reduced liver tropism, and hence toxicity compared with unmodified virus, while maintaining reporter gene expression in the tumor. The data suggest that YESIKVAVS-retargeted viruses have potential for systemic delivery for the treatment of metastatic disease.

Use of synthetic vectors for neutralising antibody resistant delivery of replicating adenovirus DNA.

Use of synthetic vectors to deliver genomes of conditionally replicating lytic viruses combines the strengths of viral and non-viral approaches by enabling neutralising antibody resistant deployment of cancer virotherapy. Adenovirus is particularly suitable for this application since all proteins essential for replication can be expressed from the input DNA, although the presence of terminal protein (TP) covalently linked to the 5' termini of the input virus genomes both improves expression of transgenes encoded in the input DNA and also enhances replication. These roles of TP were distinguished in experiments where E1-deleted Ad(GFP)DNA bearing TP (Ad(GFP)DNA-TP), delivered with DOTAP, gave a two-fold greater frequency of transduction than Ad(GFP)DNA(without TP) in non-complementing A549 cells, while in 293 cells (which support replication of E1-deleted viruses) the presence of TP mediated a much greater differential transgene expression, commensurate with its ability to promote replication. Subsequent studies using AdDNA for virotherapy, therefore, included covalently linked TP. AdDNA-TP delivered to A549 cells using a synthetic polyplex vector was shown to be resistant to levels of neutralising antisera that completely ablated infection by wild-type adenovirus, enabling polyplex/Ad(wild type)DNA-TP to mediate a powerful cytopathic effect. Similarly in vivo, direct injection of a polyplex/Ad(wild type)DNA-TP into A549 tumours was neutralising antibody-resistant and enabled virus replication, whereas intact virus was neutralised by the antibody and failed to infect. The delivery of adenovirus genomes-TP using synthetic vectors should provide a strategy to bypass neutralising antibodies and facilitate clinical application of replicating adenovirus for cancer virotherapy.

Extended plasma circulation time and decreased toxicity of polymer-coated adenovirus.

Systemic delivery of adenoviral vectors is a major goal in cancer gene therapy, but is currently prohibited by rapid hepatic uptake of virus following intravenous injection with levels of viable virus in the murine plasma typically falling to less than 0.1% after 30 min. We have used a surface-masking technique based on multivalent copolymers of poly(N-(2-hydroxypropyl)methacrylamide) to ablate all pathways of receptor-mediated infection, combined with dose modulation to achieve partial saturation of nonspecific uptake pathways. Polymer coating gave at least 100-fold decreased hepatic transgene expression at all doses and even high doses of coated virus (pc-virus) showed no weight loss or stimulation of serum transaminases. Low doses of virus and pc-virus (10(9) viral particles (vp)/mouse) were mainly captured by the liver (assessed by quantitative PCR), although higher doses led to greater fractional persistence in the plasma (measured after 30 min). Coated virus at a dose of 6 x 10(11) vp/mouse showed nearly 50% plasma circulation, representing a 3.5-fold greater area under the concentration-time curve (0-30 min) compared to unmodified virus. Such an increase in the bioavailability of adenovirus, coupled with substantial decreases in toxicity and unwanted transgene expression is an important step towards producing systemically available tumour-targeted viruses.

Enhanced efficacy of Escherichia coli nitroreductase/CB1954 prodrug activation gene therapy using an E1B-55K-deleted oncolytic adenovirus vector.

Viruses that replicate selectively in cancer cells constitute an exciting new class of anticancer agent. The conditionally replicating adenovirus (CRAd) dl1520, which lacks the E1B-55K gene, has elicited significant clinical responses in humans when used in combination with chemotherapy. A convergent development has been to use replication-defective viruses to express prodrug-activating enzymes in cancer cells. This can sensitize the cancer to prodrug, but depends upon achieving sufficient level, distribution and specificity of enzyme expression within the tumour. In this study, we have expressed the prodrug-activating enzyme nitroreductase (NTR) in the context of an E1B-55K-deleted adenovirus, CRAd-NTR(PS1217H6). We show that CRAd-NTR(PS1217H6) retains oncolytic growth properties, and expresses substantially more NTR than a comparable, replication-defective adenovirus. The combination of viral oncolysis and NTR expression results in significantly greater sensitization of SW480 and WiDr colorectal cancer cells to the prodrug CB1954 in vitro. In vivo, CRAd-NTR(PS1217H6) was shown to replicate in subcutaneous SW480 tumour xenografts in immunodeficient mice, resulting in more NTR expression and greater sensitization to CB1954 than with replication-defective virus. Combination therapy of CRAd-NTR(PS1217H6) with CB1954 reduced tumour growth from 13.5- to 2.8-fold over 5 weeks, and extended median survival from 42 to 81 days, compared with no treatment.

In vivo gene therapy for colon cancer using adenovirus-mediated, transfer of the fusion gene cytosine deaminase and uracil phosphoribosyltransferase.

Virus-directed enzyme prodrug therapy (VDEPT) utilising cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) into the chemotherapy agent, 5-fluorouracil (5-FU), and has entered into a clinical trial for metastatic colon cancer. To improve this system, a replication-deficient adenovirus, containing a bifunctional fusion gene, CD:uracil phosphoribosyltransferase (UPRT), was constructed (AdCDUPRT). UPRT enhances the conversion of 5-FU into its active metabolites, which inhibit DNA and RNA synthesis. In vitro, AdCDUPRT infection of colon cancer cells resulted in a marked increase in sensitisation to 5-FU, compared with AdCD-infected or uninfected cells. The corollary is a approximately 100-fold and approximately 10 000-fold increase in sensitisation to 5-FC in AdCDUPRT-infected cells, compared to AdCD-infected and uninfected cells, respectively. There was a strong bystander effect in vitro, 70% of tumour cells were killed by 5-FC when only 10% of cells expressed CDUPRT. In vivo, athymic mice with colon cancer xenografts treated with intratumoral AdCDUPRT and intraperitoneal 5-FC, significantly reduced tumour growth rates compared with untreated controls (P = 0.02), whereas AdCD/5-FC treated mice did not. At higher AdCDUPRT virus doses, 5-FC and 5-FU were equally effective at delaying tumour growth compared with controls. In summary, VDEPT for colon cancer utilising AdCDUPRT is more effective than AdCD and the bifunctional CDUPRT gene enables the use of either 5-FC or 5-FU as prodrugs.

Polymer-coated adenovirus permits efficient retargeting and evades neutralising antibodies.

Adenovirus is a widely used vector for cancer gene therapy because of its high infection efficiency and capacity for transgene expression in both dividing and nondividing cells. However, neutralisation of adenovirus by pre-existing antibodies can lead to inefficient delivery, and the wide tissue distribution of the coxsackie and adenovirus receptor (CAR, the primary receptor for adenovirus type 5) precludes target selectivity. These limitations have largely restricted therapeutic use of adenovirus to local or direct administration. A successful viral gene therapy vector would be protected from neutralising antibodies and exhibit a preferential tropism for target cells. We report here the development of a covalent coating and retargeting strategy using a multivalent hydrophilic polymer based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA). Incorporation of targeting ligands such as basic fibroblast growth factor and vascular endothelial growth factor on to the polymer-coated virus produces ligand-mediated, CAR-independent binding and uptake into cells bearing appropriate receptors. Retargeted virus is resistant to antibody neutralisation and can infect receptor-positive target cells selectively in mixed culture, and also in xenografts in vivo. Multivalent polymeric modification of adenovirus is an effective way of changing its tropism and interaction with the immune system. As a non-genetic one-step process, the technology is simple, versatile and should yield vectors with an improved safety profile.

Sensitisation of human carcinoma cells to the prodrug CB1954 by adenovirus vector-mediated expression of E. coli nitroreductase.

The enzyme nitroreductase from E. coli can reduce the weak, monofunctional alkylating agent 5-(aziridin-1-yl)-2, 4-dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA. Nitroreductase therefore has potential as a "suicide enzyme" for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954. We have incorporated a nitroreductase expression cassette into a replication-defective adenovirus vector (Ad-CMV-ntr), which allowed efficient gene transfer to SK-OV-3 or IGROV-1 ovarian carcinoma cells. Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx. 2, 000-fold sensitisation) with 25-50 p.f.u. per cell. Similar Ad-CMV-ntr-dependent sensitisation to CB1954 was seen in 3 of 6 low-passage primary ovarian tumour lines. Cells grown at low-serum concentration to inhibit proliferation remained equally susceptible to the Ad-CMV-ntr-dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme-prodrug systems for human tumours with a low rate of cell proliferation. Additionally, cisplatin-resistant cells were sensitised towards CB1954 by Ad-CMV-ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin-resistant tumours. In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad-CMV-ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001).

Virus-directed enzyme prodrug therapy using CB1954.

The virus-directed enzyme prodrug therapy (VDEPT) anti-cancer 'gene therapy' strategy relies on the use of viral vectors for the efficient delivery to tumour cells of a 'suicide gene' encoding an enzyme which converts a non-toxic prodrug to a cytotoxic agent. The prodrug 5-(aziridin-1-yl)-2,4 dinitrobenzamide, CB1954, has been proposed for use in enzyme-prodrug gene therapy systems with the Escherichia coli enzyme nitroreductase (Ntr). Ntr converts CB1954 to 2- and 4-hydroxylamino derivatives, whereupon the non-enzymatic reaction of the 4-hydroxylamino derivative with cellular thio- esters generates a potent cytotoxic bifunctional alkylating agent capable of cross-linking DNA. Ntr delivery has been achieved in vitro using retroviral and adenoviral vectors and confirmed by immunocytochemical demonstration of Ntr expression. The Ntr-expressing cells have been shown to be sensitized to CB1954 by up to 2000-fold. The Ntr-CB1954 system shows effective bystander killing in mixed populations of Ntr-expressing and non-expressing cells treated with CB1954. The efficacy of this enzyme-prodrug approach in model systems compared with other VDEPT approaches demonstrates the feasibility and future promise of this gene therapy strategy.

Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E. coli nitroreductase and CB1954.

Expression of the E. coli enzyme nitroreductase (NTR) in tumour cells enables them to activate the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide), leading to interstrand DNA crosslinking and cell death. Using transfected or retrovirally transduced SKOV3 ovarian carcinoma cell clones, we show a strong correlation between sensitivity to CB1954 and level of NTR enzyme activity. Importantly for clinical application in ovarian cancer, a cisplatin-resistant ovarian tumour cell line remains as susceptible to the NTR-dependent cytotoxicity of CB1954 as parental cells. In mixed populations of NTR-expressing and non-expressing cells, we observe a marked 'bystander killing' effect with this system. The use of NTR-encoding retroviruses from clonal producer cell lines at titres of 5 x 10(5) c.f.u./ml to transduce either established or low passage primary ovarian carcinoma lines only achieves an average 10-fold sensitisation of the cultures at gene transfer efficiencies of 15-25%. Concentration of the retrovirus to 3 x 10(7) c.f.u./ml elevates gene transfer to 80-90% in a single exposure to target cells, resulting in up to 500-fold sensitisation of the entire, unselected SKOV3 population to CB1954. In an initial investigation of NTR/CB1954 for the treatment of tumours in vivo, we observe regression of tumours expressing NTR following administration of CB1954, resulting in significantly increased median survival.

Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E. coli nitroreductase gene.

Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy. CB1954 is a weak, monofunctional alkylating agent which can be activated by Escherichia coli nitroreductase to a potent dysfunctional alkylating agent which crosslinks DNA. We have inserted the nitroreductase gene into an LNCX-based retroviral vector, to allow efficient gene transfer and expression in colorectal (LS174T) and pancreatic (SUIT2, BxPC3, and AsPC1) cancer cell lines. A clone of LS174T cells expressing nitroreductase showed > 50-fold increased sensitivity to CB1954, and nitroreductase-expressing clones of pancreatic tumor lines were up to approximately 500-fold (SUIT2) more sensitive than parental cells. Concentrations of CB1954 minimally toxic to nontransduced cells achieved 100% cell death in a 50:50 mix of parental cells with SUIT2 cells expressing nitroreductase; and marked "bystander" cell killing was seen with just 10% of cells expressing nitroreductase. Significant bystander cell killing was dependent on a high cell density. In conjunction with regional delivery of vectors and tumor selectivity of cell entry and/or gene expression, nitroreductase and CB1954 may be an attractive combination for prodrug-activating enzyme gene therapy of colorectal and pancreatic cancer.

Transfection of cardiac muscle: effects of overexpression of c-myc and c-fos proto-oncogene proteins in primary cultures of neonatal rat cardiac myocytes.

1. Studies of cells transfected with specific expression vectors allow functions of specific gene products to be investigated. We first established optimum conditions for transfection of neonatal cardiac myocytes in primary culture. Increased proto-oncogene expression has been implicated in the development of cardiac hypertrophy; we therefore wished to examine the effects of overexpression of the c-myc and c-fos proto-oncogenes induced by cell transfection on cell growth. 2. Neonatal rat ventricular myocytes were transfected by electroporation, voltage and capacitance settings were optimized and these conditions were then used to transfect expression vectors encoding MYC and FOS proteins into this cell type. Effects on cell number, DNA synthesis and protein content were determined. 3. Increased expression of c-myc and c-fos in cells transfected with proto-oncogene expression vectors was confirmed by Northern and Western blotting. Increases in cardiac myocyte cell number, DNA synthesis and total cellular protein were observed in cells transfected with either c-myc or c-fos expression vectors compared with cells transfected with non-coding vectors. 4. Overexpression of MYC and FOS proteins results in hyperplastic rather than hypertrophic growth of the neonatal rat heart, providing evidence for a role of these proteins in the control of myocardial growth.

Regulation of beta myosin heavy chain, c-myc and c-fos proto-oncogenes in thyroid hormone-induced hypertrophy of the rat myocardium.

1. In order to investigate the molecular mechanisms determining the hypertrophic response of the ventricular myocardium to thyroid hormone administration, changes in left and right ventricular expression of the c-myc, c-fos and H-ras proto-oncogenes in response to treatment with 3,3',5-tri-iodothyronine were defined. 2. Adult female Wistar rats were treated with daily subcutaneous injections of 3,3',5-tri-iodothyronine (50 micrograms) for 1, 3, 7 or 14 days (n = 6 in each treatment group) and the results from 3,3',5-tri-iodothyronine-treated animals were compared with those obtained from untreated controls (n = 6). Changes in the weight of the left and right ventricles in response to 3,3',5-tri-iodothyronine treatment were measured; changes in expression of the c-myc, c-fos and H-ras proto-oncogenes were determined in parallel by measurement of specific messenger RNAs by Northern and dot hybridization, as well as changes in expression of beta myosin heavy chain messenger RNA. 3. Treatment with 3,3',5-tri-iodothyronine resulted in increases in both left and right ventricular weights after 3 days, an effect maintained up to 14 days. Despite an increase in left ventricular weight, levels of beta myosin heavy chain, c-myc, c-fos and H-ras mRNAs in the left ventricle were unchanged; in contrast, an increase in right ventricular weight was associated with increased expression of beta myosin heavy chain, c-myc and c-fos messenger RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of experimental hypertension on phosphoinositide hydrolysis and proto-oncogene expression in cardiovascular tissues.

Products of inositol lipid hydrolysis and levels of c-myc, c-fos and H-ras mRNAs were measured in rat left ventricle and vascular tissues 72 h and 9 days after the induction of aortic coarctation in order to examine inositol phosphate and proto-oncogene signals during the development of pressure-related cardiac and vascular structural changes. There was a significant increase in left ventricular and proximal aortic mass at both time points but no change in mesenteric resistance artery morphology in rats with coarctation. At 72 h there was a significant increase in c-myc, c-fos and H-ras mRNAs in the left ventricle of rats with coarctation, and this was accompanied by increased levels of inositol (1,4,5)-trisphosphate. Similar results were obtained in the proximal but not the distal aorta. In resistance arteries inositol phosphate production and proto-oncogene mRNA expression were unchanged. The results indicate that at 72 h aortic coarctation induced structural thickening in the left ventricle and proximal aorta and was associated with increased inositol phosphate production and stimulation of specific proto-oncogene mRNAs. By 9 days following surgery much of the structural change in these tissues was completed, and these raised cellular signals were no longer observed. The results suggest that both increased inositol lipid hydrolysis and a rise in the expression of these proto-oncogenes are important processes in the development of vascular hypertrophy seen in this model of hypertension.

Regulation by thyroid status of c-myc, c-fos and H-ras mRNAs in the rat myocardium.

Effects of thyroid status on expression of a variety of myocardial genes, such as those encoding contractile proteins, have been reported, as well as interactions between thyroid hormones and developmental and haemodynamic regulation of contractile protein synthesis. In addition, it is clear that developmental and haemodynamic factors regulate expression of specific proto-oncogenes, including c-myc, c-fos and H-ras, in the myocardium but the effect of thyroid status on such proto-oncogene products, which are proposed to play a critical signal-transducing role in the heart, has been previously unexplored. In order to determine whether changes in thyroid status are associated with changes in expression of these putative intracellular signals, we examined the effect of hypothyroidism and tri-iodothyronine (T3) treatment on myocardial levels of c-myc, c-fos and H-ras mRNAs in the rat. The induction of hypothyroidism was associated with a marked increase in myocardial c-myc, c-fos and H-ras mRNAs, changes reversed by 72 h of T3 replacement. Administration of T3 to euthyroid rats had no significant effect on myocardial c-myc or c-fos mRNAs, but inhibition of H-ras mRNA by T3 was evident. These observations demonstrating influences of thyroid status on expression of specific proto-oncogenes suggest that thyroid hormones, as well as exerting direct effects on expression of functionally important myocardial genes, also interact with the cellular transduction pathways mediated by the products of the c-myc, c-fos and H-ras genes.

Differential regulation by thyroid hormones of myosin heavy chain alpha and beta mRNAs in the rat ventricular myocardium.

The effect of tri-iodothyronine (T3) treatment on myocardial levels of alpha and beta myosin heavy chain (MHC) mRNAs in the rat was defined in vivo and in vitro. Dose-response experiments were performed in intact hypothyroid and euthyroid rats; in addition, studies in vitro examined the effect of T3 on MHC mRNAs in neonatal cardiac myocytes in primary culture. Specific alpha and beta MHC mRNAs were determined by Northern blot and dot hybridization to oligonucleotide probes complementary to the 3' untranslated regions of the MHC genes. An increase in myocardial beta MHC mRNA was demonstrated in hypothyroidism, accompanied by a reduction in alpha MHC mRNA. Marked differences in the sensitivity of alpha and beta MHC mRNAs to T3 replacement were found; a dose-dependent increase in alpha mRNA was evident at 6 h after T3 treatment, in the absence of consistent effects on beta mRNA, whereas 72 h after T3 replacement was commenced, stimulatory effects of T3 on alpha MHC mRNA, evident at all doses, were accompanied by a dose-dependent inhibition of beta MHC mRNA. No effect of thyroid status on actin mRNA was found, indicating the specificity of MHC gene regulation. T3 treatment of cardiac myocytes in vitro exerted similar actions on MHC mRNAs to those found in vivo, with a more marked influence on alpha than beta MHC mRNA. These studies of the action of T3 in vivo and in vitro have thus demonstrated specific effects of T3 on pretranslational regulation of the alpha and beta MHC genes, influences which differ not only in terms of stimulation or inhibition, but also in magnitude of effect.

Regulation of alpha- and beta-myosin heavy chain messenger RNAs in the rat myocardium by amiodarone and by thyroid status.

1. A number of the cardiovascular effects of amiodarone resemble those of hypothyroidism, prompting examination of the relationship between the actions of the drug and thyroid hormones. Amiodarone treatment of the rat was used as a model to determine the influence of the drug on thyroid hormone-regulated gene expression in the myocardium and liver; interactions between amiodarone and thyroid status were examined in hypothyroid and tri-iodothyronine (T3)-treated animals. 2. Myocardial levels of alpha- and beta-myosin heavy chain (MHC) messenger RNAs (mRNAs) were measured by dot hybridization to specific oligonucleotide probes; myocardial actin mRNA was measured in parallel by hybridization to a complementary DNA (cDNA) probe. Hepatic levels of Spot 14 and thyroxine-binding prealbumin mRNA were similarly determined by dot hybridization to radiolabelled cDNAs. 3. Amiodarone treatment of the rat resulted in specific increases in both alpha- and beta-MHC mRNAs in the myocardium, as well as hepatic Spot 14 mRNA, changes reversed by T3 administration. 4. Hypothyroidism resulted in a reduction in myocardial alpha-MHC and hepatic Spot 14 mRNAs, in contrast to amiodarone, whilst hypothyroidism and amiodarone both exerted stimulatory influences on beta-MHC mRNA. Treatment of hypothyroid rats with amiodarone had no significant effect on beta-MHC or Spot 14 mRNAs, but a further reduction in alpha-MHC mRNA, compared with the untreated hypothyroid state, was evident. 5. The demonstrated influence of amiodarone on both alpha- and beta-MHC mRNAs and interactions between amiodarone and thyroid status in regulating MHC gene expression may be relevant to its therapeutic effect in man.

The chloroplast-located glutamine synthetase of Phaseolus vulgaris L.: nucleotide sequence, expression in different organs and uptake into isolated chloroplasts.

Work using a full-length cDNA clone has revealed that the plastid-located glutamine synthetase (GS) of Phaseolus vulgaris is encoded by a single nuclear gene. Nucleotide sequencing has shown that this cDNA is more closely related to a cDNA encoding the plastidic GS of Pisum sativum than to cDNAs encoding three different cytosolic GS subunits of P. vulgaris. The plastid GS subunits are initially synthesized as higher M r (47000) precursors containing an N-terminal presequence of about 50 amino acids which is structurally similar to the presequences of other nuclear-encoded chloroplast proteins. The precursor has been synthesized in vitro and is imported by isolated pea chloroplasts and processed to two polypeptides of the same size as native P. vulgaris chloroplast GS subunits (M r 42000). Experiments with fusion proteins show that the N-terminal 68 amino acids of this precursor allow the cytosolic GS subunit ß also to be imported and processed by isolated chloroplasts. Polyadenylated mRNA specifically related to the plastidic GS gene is most highly abundant in chloroplast-containing organs (leaves and stems) but is also detectable in roots and nodules.