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Abstract
Sequences for human S-adenosylmethionine decarboxylase, an enzyme involved in polyamine biosynthesis, which is elevated in tumors, have been localized on chromosome 6 and the X.
Increased polyamine biosynthetic activity and raised levels of intra-cellular polyamines are commonly associated with normal, abnormal, and induced proliferative states. Ornithine Decarboxylase and S-adenosylmethionine Decarboxylase are known rate-limiting enzymes in polyamine biosynthesis. Herein, we report results from our group in relation to the activity of these enzymes in colonic neoplasias and their possible role as tumor markers.
Increased polyamine biosynthetic activity and raised levels of intracellular polyamines are commonly associated with abnormal and induced proliferative states. Ornithine Decarboxylase and S-Adenosylmethionine Decarboxylase are the known rate-limiting enzymes in polyamine biosynthesis. Herein, we report results from our group in relation to the activity of these enzymes in “normal looking” mucosa and neoplasias of the colon. Both enzymes tend to reflect neoplastic progression (mucosa<polyp < carcinoma). During these studies, we observed low levels of activity in the “normal looking” mucosa of colons containing a neoplasia, but high levels of ODC in “normal looking” mucosa of the same colons after the removal of their neoplasias. These observations suggest a role of ODC as a possible biochemical marker of a colon harboring a neoplasia.
Genomic clones for the S-adenosylmethionine (AdoMet) decarboxylase gene were isolated from a human chromosome 6 DNA library. In addition, polymerase chain reaction and specific primers were used to amplify fragments from chromosomal DNA covering exonic regions not found in the screening of DNA libraries with AdoMet decarboxylase cDNA. The gene encompasses at least 22 kilobases of chromosome 6 DNA and comprises nine exons and eight introns, in contrast to the corresponding rat gene that has only eight exons (Pulkka, A., Ihalainen, R., Aatsinki, J., and Pajunen, A. (1991) FEBS Lett. 291, 289-295). Exon-intron junctions in the human and rat AdoMet decarboxylase genes were in identical positions except that exons 6 and 7 of the human gene formed a single exon in the rat gene. Alu-like sequences are present in four introns and the 5'-flanking region of the human gene. The promoter region contains a TATA box adjacent to the cap site; in addition, DNA elements for binding of transcription factors AP-1, AP-2, CREB, SP-1, and multiple steroid receptors are present between position -3,158 and the transcription start site. Two AdoMet decarboxylase promoter-reporter gene constructs with about 170 and 1,500 nucleotides of the 5'-flanking DNA were used in transient expression studies. AdoMet decarboxylase promoter was capable of driving reporter gene expression, but it was less active than the murine ornithine decarboxylase promoter. There are at least three potential polyadenylation signals at the 3'-end of the gene, and utilization of the first two results in the formation of the 2.0- and 3.6-kilobase AdoMet decarboxylase mRNA species present in human tissues and cell lines. AdoMet decarboxylase gene-related sequences were also present in a human X chromosome-specific DNA library. Partial nucleotide sequencing of this DNA revealed a lack of introns present in the gene located on chromosome 6, suggesting that the locus on the X chromosome contains a processed AdoMet decarboxylase pseudogene.
The polyamine biosynthetic pathway has attracted much interest as a therapeutic target. Many studies have shown the potential value of inhibitors of the first enzyme in the biosynthetic pathway, ornithine decarboxylase, which forms putrescine. In order to convert putrescine into the polyamines, spermidine and spermine, the aminopropyl donor, decarboxylated S-adenosylmethionine, is needed. Therefore, S-adenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50) is essential for polyamine synthesis. Early studies of the inhibition of this enzyme were carried out with compounds such as methylglyoxal bis(guanylhydrazone) that lack specificity and also lack potency since they are competitive inhibitors whose effects are overcome by a compensatory increase in the amount of the target enzyme. Recently, powerful irreversible inhibitors of AdoMetDC have become available including 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine, an enzyme activated inhibitor and 5'-deoxy-5'-[(3-hydrazinopropyl)methylamino]adenosine which binds to the active site and forms a covalent bond with the pyruvate prosthetic group. This review describes the current state of knowledge of the structure and properties of AdoMetDC, the available inhibitors of this enzyme, their mechanism of action and their effects on polyamines and on the growth of tumors and protozoan parasites. These effects indicate that AdoMetDC inhibitors may be of therapeutic value either alone or in combination with ornithine decarboxylase inhibitors and that further trials of these compounds should be considered.
The nucleotide sequence of a cDNA encoding the proenzyme of hamster S-adenosylmethionine decarboxylase including 169 nucleotides of the 5' untranslated region has been determined. The deduced amino acid sequence shows a remarkable similarity to the human proenzyme with only seven differences out of 334 amino acids. The nucleotide sequence of the 5' untranslated region showed 93% homology with the corresponding rat and human sequences suggesting that this region may play an important role in the regulation of S-adenosylmethionine decarboxylase expression.
The polyamines are known to be essential for cellular proliferation. Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the synthesis of these amines, and activity is elevated in colorectal tumors and polyps. Two ODC genes (designated ODC1 and ODC2) were localized by somatic cell hybridization and in situ techniques to 2p25 and 7q31-qter, respectively. Investigation of the expression of ODC in colorectal neoplasia reveals a consistent increase in mRNA expression compared with normal adjacent mucosa and control mucosa, ranging from 1.3- to 12.2-fold. No amplification of the loci was seen. Comparison of ODC mRNA expression with ODC activity from the same samples revealed no direct correlation, suggesting that regulation of ODC in this system occurs at the posttranscriptional level.
The human spermidine synthase (EC 2.5.1.16) gene was isolated from a genomic library constructed with DNA obtained from a human immunoglobulin G (IgG) myeloma cell line. Subsequent sequence analyses revealed that the gene comprised of 5,818 nucleotides from the cap site to the last A of the putative polyadenylation signal with 8 exons and 7 intervening sequences. The 5'-flanking region of the gene was extremely GC rich, lacking any TATA box but containing CCAAT consensus sequences. No perfect consensus sequence for the cAMP-responsive element for the AP-1 binding site was found, yet the gene contained seven AP-2 binding site consensus sequences. The putative polyadenylation signal was an unusual AATACA instead of AATAAA. Polymerase chain reaction analysis with DNA obtained from human x hamster somatic cell hybrids indicated that human spermidine synthase genomic sequences segregate with human chromosome 1. Transfection of the genomic clone into Chinese hamster ovary cells displaying a low endogenous spermidine synthase activity revealed that the gene was transiently expressed and hence in all likelihood represents a functional gene.
The polyamines putrescine, spermidine and spermine are important cellular constituents involved in the regulation of cell growth and differentiation. Their intracellular levels are regulated by a multitude of mechanisms affecting their synthesis, degradation, uptake and excretion. As a result of the application of molecular biology techniques, some of these mechanisms are presently being unravelled, and are providing a basis for the rational development of novel agents effective against proliferative disorders and various parasitic diseases.
S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Its product, decarboxylated S-adenosylmethionine (dcAdoMet) is used as an aminopropyl donor by spermidine synthase and spermine synthase (Williams-Ashman and Pegg, 1981; Pegg and McCann, 1982; Tabor and Tabor, 1984a). Once decarboxylated by the action of AdoMetDC, S-adenosylmethionine (AdoMet) becomes committed to polyamine production since methyltransferases use dcAdoMet very poorly, if at all (Pegg, 1984, 1986). In fact, the only known metabolic route for further metabolism of dcAdoMet apart for its use as an aminopropyltransferase substrate is its acetylation (Wagner et al., 1985; Pegg et al., 1986) The supply of dcAdoMet is normally regulated very tightly by the cellular polyamine content and this regulation is brought about by changes in the activity of AdoMetDC (Pegg, 1984). In this way, the cellular content of dcAdoMet is usually kept very low (about 1–3% of AdoMet content) as its synthesis is linked to the ability of the aminopropyltransferases to use it to form polyamines. Only when cellular polyamine metabolism is deranged by inhibition of the other enzymes in the polyamine biosynthetic pathway does the dcAdoMet content rise. Increases of several hundred fold occur when ornithine decarboxylase (ODC) activity is inhibited by drugs such as α-difluoromethylornithine (DFMO) and, only under these conditions, is the acetyl derivative of dcAdoMet formed in significant amounts (Pegg, 1986). The increased content of dcAdoMet is due to both an increase in the activity of AdoMetDC (Alhonen-Hongisto, 1980; Mamont et al., 1981; Pegg, 1984) and to the inability of the aminopropyltransferases to utilize the dcAdoMet formed by it because of the absence of putrescine and spermidine to serve as aminopropyl acceptors.
With the use of the isoschizomeric restriction endonucleases HpaII and MspI, we found that mouse tumour ornithine decarboxylase (ODC; EC 4.1.1.17) genes are extensively methylated. ODC genes in L1210 mouse leukaemia cells were apparently more methylated than in Ehrlich ascites carcinoma, as revealed by the use of HpaII endonuclease, yet the digestion of genomic DNA isolated from these two murine tumour cell lines with MspI, which cleaves at a CCGG sequence, also with internally methylated cytosine, resulted in an apparently identical restriction pattern. It is possible that the amplification of ODC genes in Ehrlich ascites-carcinoma cells in response to 2-difluoromethylornithine (DFMO) was associated with hypomethylation, or that less-methylated genes were amplified. A human myeloma (Sultan) cell line only revealed three separate hybridization signals when cleaved with HpaII. One of these signals was amplified under the pressure of DFMO. When cleaved with MspI, these three HpaII fragments disappeared and were replaced by a double signal of 2.3-2.4 kilobase-pairs (kbp) in size. The amplified ODC sequences in the Sultan myeloma cell line apparently originated from chromosome 2, as indicated by a unique hybridization signal in a 5.8 kbp HindIII fragment specific for the human ODC locus on chromosome 2. A comparison of different human cells, the Sultan myeloma, a lymphocytic B-cell leukaemia (Ball), normal mononuclear leucocytes and leucocytes obtained from leukaemia patients, revealed interesting differences in the methylation of ODC genes. The use of two restriction endonucleases (HpaII and CfoI), the cleavage site for both of which contains a CG sequence and which only cleave when cytosine is unmethylated, indicated that ODC genes in the lymphocytic leukaemia cells were much less methylated than those in the normal leucocytes or in the Sultan cells.
Analogues of S-adenosylmethionine with modifications in the 5′-group were prepared as potential inhibitors of S-adenosylmethionine decarboxylase (AdoMet-DC). These new analogues contained carbonyl-reactive end groups in the 5′-side chain, designed to interact favorably with the pyruvate prosthetic group of AdoMet-DC. Several of the analogues proved to be outstanding inhibitors of the enzyme. The analogues were also evaluated for their activity against human cytomegalovirus in vitro.
Polyamine biosynthetic activity was assessed in various colorectal tissue samples consisting of noninvolved mucosa, benign adenomatous polyps and adenocarcinomas taken at surgery from a total of 40 patients. Ornithine decarboxylase (ODC) displayed a gradient of enzyme activity (i.e., adenocarcinoma > polyps > mucosa) which seemed to correlate positively with the neoplastic status of the tissue. In 10 of the patients, samples were obtained for all three tissue types. Five of these exhibited a clear repetition of the trends in enzyme activity seen with the mixed patient tissue sampling whereas the remainder differed by having the highest ODC activity in the polyps. In nine of the ten cases, ODC activity was substantially lower in the mucosa than in either of the neoplastic lesions. Trends in enzyme activity were the same for tissues obtained from either the colon or rectum. The ODC activity in adenocarcinomas could not be correlated with histologic differentiation, stage or site of the disease, however, in samples from female patients (all postmenopausal) the activity was elevated over normal mucosa to a greater extent (ten-fold) than in male patients (seven-fold). S-adenosylmethionine decarboxylase activity was assessed in 27 of the 40 patients and found to follow the same distribution as ODC; however, the mean value differences ± SEM between tissues were less distinct. In general, tissue polyamine pool analysis of these same specimens reflected the levels of ornithine and S-adenosylmethionine decarboxylase activities. Overall, the data reveal an increase in polyamine biosynthetic activity in colorectal neoplasms, relative to surrounding mucosa, which may correlate with (1) progression of the neoplastic process, (2) the proportion of proliferating cells, (3) the rate of cell proliferation, or (4) a combination of two or all of these possibilities.
Although ornithine decarboxylase under most conditions is the rate-controlling enzyme of polyamine biosynthesis and thus the most logical target for chemical intervention, the inhibition of the enzyme triggers a series of compensatory reactions all aimed to circumvent the inhibition. These include secondary induction of adenosylmethionine decarboxylase, enhanced accumulation of extracellular polyamines and an overproduction of ornithine decarboxylase resulting from enhanced expression and gene amplification. Thus chemotherapy based on an intervention of polyamine formation has also to be directed to reactions other than the decarboxylation of ornithine. Adenosylmethionine decarboxylase is the second natural target for chemotherapy. Virtually all effective inhibitors of this enzyme are members of the family of bis(guanylhydrazones). Small modifications, such as increased hydrophobicity at the glyoxal portion of the parent compound glyoxal bis(guanylhydrazone), greatly enhance the inhibition of adenosylmethionine decarboxylase and diminish the undesirable inhibition of diamine oxidase. However, although ethylglyoxal and propyglyoxal bis(guanylhydrazone) appear to utilize the putative polyamine carrier for their cellular entry, their cellular accumulation, in contrast to that of glyoxal and methylglyoxal bis(guanylhydrazone), is not stimulated by putrescine and spermidine deprivation produced by inhibitors of ornithine decarboxylase. It is obvious that the cellular accumulation of each of the bis(guanylhydrazones) is determined by their different efflux rates: GBG and MGBG are effectively retained whereas EGBG is rapidly excreted by the tumor cells. GBG and MGBG, but possibly not EGBG, behave as mitochondrial poisons and rapidly produce extensive morphological damage of the mitochondria. The bis(guanylhydrazones) likewise inhibit carnitine-dependent mitochondrial oxidation of long-chain fatty acids, competitively in respect to carnitine. It is possible that this inhibition has something to do with the mitochondrial damage, as carnitine protects tumor cells from the early mitochondrial damage produced by MGBG. Carnitine also protects experimental animals from MGBG-induced acute toxicity and death.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
Analogues of S-adenosylmethionine that were designed as inhibitors of S-adenosylmethionine decarboxylase were tested for their abilities to inhibit the purified enzyme from rat prostate. The most potent inhibitors were 5'-deoxy-5'-[N-methyl-N-[2-(aminooxy)ethyl]amino]adenosine (MAOEA) and 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine (MHZPA), which had I50 values of 400 nM and 70 nM, respectively, when added directly to the assay medium under standard conditions. These compounds were irreversible inactivators of the enzyme, and more than 95% of the activity was lost within 15 min of exposure to 5 microM MAOEA or 0.5 microM MHZPA. Both inhibitors led to a large reduction in the content of decarboxylated S-adenosylmethionine in L1210 cells and to a substantial decrease in the production of 5'-(methylthio)adenosine by these cells. These results are consistent with their bringing about an inhibition of S-adenosylmethionine decarboxylase activity in the cell which leads to a reduction in the synthesis of spermidine and spermine. Analysis of the polyamine content in L1210 cells exposed to 100 microM MAOEA or 50 microM MHZPA showed that this was the case and that putrescine levels were greatly increased while spermidine and spermine content declined. The combined application of 100 microM MAOEA and 5 mM alpha-(difluoromethyl)ornithine (an ornithine decarboxylase inhibitor) to L1210 cells completely prevented the synthesis of putrescine, spermidine, and spermine for up to 48 h. The reduction in polyamine content brought about by MHZPA or MAOEA could be partially prevented by the addition of decarboxylated S-adenosylmethionine to the culture medium. These inhibitors also brought about an inhibition of cell growth which could be reversed by the addition of spermidine.(ABSTRACT TRUNCATED AT 250 WORDS)
A cloned human cDNA probe for fibronectin (FN) containing 1.3 kb of the human FN coding region has been used to determine the chromosome that encodes the structural gene in human-mouse somatic cell hybrids. The results show that human chromosome 2 encodes the FN structural gene.
This report deals with acquired recurring clonal structural chromosome abnormalities in neoplasia. They are confined to the neoplastic tissue. The definition of a clone in this context is the presence of an identical structural chromosome change in at least 2 cells. The results are presented by chromosome in Table I, and by disease in Table II. The chromosomal localizations of known proto-oncogenes are listed in Table III. As recommended at HGM8, to avoid the implication that fragile sites are related to neoplasia, they are no longer included in the report of this committee on chromosome aberrations in neoplasia.