Biomedical subjects

C W Porter

Publications and source records attributed to C W Porter.

At least 37 records · Page 2Linked to original sources

PubMed1997

Activation of polyamine catabolism profoundly alters tissue polyamine pools and affects hair growth and female fertility in transgenic mice overexpressing spermidine/spermine N1-acetyltransferase.

We have generated a transgenic mouse line that overexpresses the rate-controlling enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase. Tissues of these mice showed markedly distorted polyamine pools, which in most cases were characterized by the appearance of N1-acetylspermidine, not normally found in mouse tissues, a massive accumulation of putrescine, and decreases in spermidine and/or spermine pools. The most striking phenotypic change was permanent hair loss at the age of 3 to 4 weeks which was typified histologically by the appearance of extensive follicular cysts in the dermis. The effect seemed attributable to putrescine interference with hair development, possibly with differentiation/proliferation of epidermal cells located in hair follicles. Female members of the transgenic line were found to be infertile apparently due to ovarian hypofunction and hypoplastic uteri. The findings demonstrate the utility of spermidine/spermine N1-acetyltransferase overexpression as an effective means for genetically modulating total tissue polyamine pools in transgenic animals and examining the developmental and oncogenic consequences.

Acetyltransferases↗

PubMed1997

Structural basis for differential induction of spermidine/spermine N1-acetyltransferase activity by novel spermine analogs.

The spermine analog N1,N11-diethylnorspermine (DE-333, also known as DENSPM or BENSPM) is regarded as the most potent known inducer of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), increasing activity by more than 200- to 1000-fold in certain cell types. The relative ability of a series of eight systematically modified DE-333 analogs to affect SSAT expression was examined in Malme-3M human melanoma cells, one of several cell lines known to be especially responsive to induction of this enzyme. In particular, we examined the relative contribution of induction of enzyme mRNA and prolongation of enzyme half-life to analog-mediated increases in enzyme activity. Induction of enzyme mRNA was most influenced by intra-amine carbon distances; relative effectiveness was found to be proportional to the number of three-carbon units. Stabilization of enzyme was most determined by the terminal N-alkyl substituent size; among methyl, ethyl and propyl groups, methyl was least effective. Thus, DE-333, which most potently induces SSAT mRNA and effectively stabilizes SSAT enzyme activity, produces the greatest increase in enzyme activity. Although other contributing mechanisms may be involved, the relative abilities of the various analogs to induce enzyme activity is at least partially attributable to their combined effects on enzyme mRNA and protein half-life. These data reveal the highly sensitive structure-activity relationships that underlie and control spermine analog induction of SSAT activity. Pending further definition of the relationship between SSAT induction and antitumor growth and toxicity in vivo, these relationships may be used to optimize therapeutic efficacy.

Acetyltransferases↗

PubMed1997

Antitumor efficacy of N1,N11-diethylnorspermine on a human bladder tumor xenograft in nude athymic mice.

The spermine analogue N1,N11-diethylnorspermine (DENSPM) has been shown to induce the polyamine-acetylating enzyme spermidine/spermine N1-acetyltransferase, disrupt polyamine pool homeostasis, and inhibit tumor growth. DENSPM is currently being developed as an anti-neoplastic agent and is about to enter Phase II clinical trials. In this report, the antitumor efficacy of DENSPM was evaluated against a human transitional cell bladder BL13 carcinoma xenograft implanted orthotopically and s.c. in nude athymic mice. DENSPM was administered via continuous s.c. infusion at 93 mg/kg/day for 5 days. Treatment with DENSPM was well tolerated and produced tumor regressions in all mice with a significant proportion (up to 50%) of apparent cures. On the basis of low toxicity and good therapeutic efficacy, there is a strong rationale for evaluation of the therapeutic efficacy of DENSPM against bladder carcinomas in Phase II clinical trials.

Animals↗

PubMed1996

Effects of polyamines, polyamine analogs, and inhibitors of protein synthesis on spermidine-spermine N1-acetyltransferase gene expression.

The key polyamine catabolizing enzyme spermidine-spermine N1-acetyltransferase (SSAT) is among the few genes known to be inducible by the natural polyamines. Certain polyamine analogs markedly exaggerate this response and thus provide useful tools for studying the underlying regulatory mechanisms. As shown here, the analog which most potently induces SSAT activity, N1, N11-diethylnorspermine (DENSPM), increases SSAT mRNA in MALME-3M human melanoma cells to a maximum of > 20-fold and immunodetectable SSAT protein to > 300-fold. By comparison, the natural polyamine spermine is far less effective, increasing SSAT mRNA by approximately 3-fold and protein by approximately 7-fold. In particular, the difference in mRNA accumulation by spermine and the analog was shown to be due to differential effects on both gene transcription and mRNA stabilization. Although the analog DENSPM has been regarded as the most potent inducer of SSAT activity and mRNA, we now report that inhibitors of protein synthesis are capable of increasing SSAT mRNA to nearly comparable levels. Inhibitor-induced accumulation in SSAT mRNA was shown to involve increased gene transcription and mRNA stabilization. This suggests that, under basal conditions, SSAT gene expression is suppressed by a labile protein (or proteins). While induction of SSAT mRNA by inhibitors of protein synthesis only occurred at concentrations which blocked protein synthesis, that by DENSPM took place at concentrations which did not. The combination of either protein inhibitor with DENSPM or spermine produced an additive increase in SSAT mRNA. Taken together, these findings suggest the involvement of two separate but possibly converging pathways in the regulation of SSAT mRNA, one mediated by polyamines and their analogs and the other mediated by a labile repressor of SSAT gene transcription and/or mRNA stabilization. In addition to its apparent regulatory importance, induction of SSAT mRNA by inhibitors of protein synthesis represents a potentially useful system for studying the posttranscriptional regulation of this interesting gene.

Acetyltransferases↗

PubMed1996

Differential post-transcriptional control of ornithine decarboxylase and spermidine-spermine N1-acetyltransferase by polyamines.

Ornithine decarboxylase (ODC) and spermidine/ spermine N1-acetyltransferase (SSAT) are short-lived polyamine enzymes with rate-limiting roles in controlling polyamine biosynthesis and catabolism, respectively. We have found that treatment of MALME-3M human melanoma cells for 6 h with 10 micrograms/ml cycloheximide (CHX) increases ODC and SSAT mRNA 6-9-fold. When cells containing CHX-induced SSAT mRNA were washed and post-incubated for an additional 6 h in drug free media, enzyme activity increased only 2-fold above that in untreated cells despite the > 6-fold increase in accumulated mRNA. Inclusion of 10 microM spermine or spermidine in the post-incubation medium increased SSAT activity approximately 7-fold without further elevating SSAT mRNA levels. This indicates post-transcriptional regulation which, due to the similarity between polyamine-mediated increases in SSAT activity and available mRNA, probably occurs at the level of mRNA translation. In contrast to the SSAT response, polyamines markedly reduced ODC activity (but not mRNA) to one sixth that in cells not exposed to polyamines. The findings illustrate how via post-transcriptional mechanisms, shifts in intracellular polyamine pools can simultaneously and differentially regulate polyamine biosynthesis and catabolism. It is hypothesized that these post-transcriptional responses enable cells to rapidly and sensitively control intracellular spermidine and spermine pools.

Acetyltransferases↗

PubMed1996

c-Myc induces apoptosis and cell cycle progression by separable, yet overlapping, pathways.

Enforced c-Myc expression promotes inappropriate cell cycle progression of growth factor deprived cells and triggers concomitant apoptosis. However, it is not clear what role dysregulation of the cell cycle plays in c-Myc-induced apoptosis. Ornithine decarboxylase (ODC) is a transcriptional target of c-Myc and contributes to c-Myc induced apoptosis. Here we have established that high levels of ODC overexpression in interleukin-3 (IL-3)-dependent 32D.3 myeloid cells induces apoptosis at rates comparable to those induced by enforced c-Myc expression. However, ODC-induced apoptosis was not accompanied by dysregulation of cell cycle controls, indicating that cell death was not triggered by inappropriate cell cycle progression. Nonetheless, ODC was required downstream of c-Myc for myeloid cell growth. These results suggested that c-Myc-induced pathways leading to cell cycle progression and apoptosis are separable, yet that they share common mediators. In agreement with this concept, treatment of cells over-expressing c-Myc with the growth inhibitory agent dibutyryl cyclic AMP (Bt2cAMP) arrested these cells G1, without inducing apoptosis. However, c-Myc retained the ability to induce apoptosis of Bt2cAMP-arrested cells following removal of IL-3, demonstrating that Bt2cAMP selectively inhibits c-Myc-induced pathways promoting cell cycle progression but not apoptosis. These results suggest a "multiple effectors' model in which c-Myc regulates the expression of mediators which alone are sufficient to induce apoptosis in the absence of survival factors, yet are required in concert to promote cell cycle progression.

Animals↗

PubMed1996

[New aminooxy analogs of biogenic polyamines].

A series of structural analogs of putrescine, spermidine, and spermine with the aminomethylene fragment substituted by the aminooxy group was suggested. The synthesis of the new aminooxy analogs of spermine was described. Biochemical aspects of the activity of the aminooxy analogs of polyamines were discussed in respect of their selective inhibition of normal and leukemic cells.

Animals↗

PubMed1995

Photoaffinity labeling of a cell surface polyamine binding protein.

Intracellular polyamine pools are partially maintained by an active transport apparatus that is specific for and regulated by polyamines. Although mammalian transport activity has been characterized by kinetic studies, the actual protein itself has yet to be identified, purified, or cloned. As one approach to this problem, we attempted photoaffinity labeling of plasma membrane proteins using two specifically designed and synthesized polyamine conjugates as photoprobes. The first is a spermidine conjugate bearing the photoreactive moiety 4-azidosalicylic acid at the N4 position via an alkyl linkage, and the second is a norspermine conjugate with 4-azidosalicylic acid at the N4 position via an acyl linkage. Labeling of murine L1210 lymphocytic leukemia cells was carried out at 4 degrees C to promote selective alkylation of cell surface proteins. Separation of plasma membrane proteins from cells cross-linked with the N4-spermidine conjugate by SDS-polyacrylamide gel electrophoresis revealed two heavily labeled proteins at approximately 118 and approximately 50 kDa (designated p118 and p50, respectively). Band p118 was more well defined and much more intensely labeled. Analogous proteins were also observed in human U937 lymphoma cells. Specificity of labeling was strongly suggested by competition with polyamines and analogs during labeling and further indicated by the nearly identical labeling of the same protein by the N1-norspermine photoprobe but not by the unconjugated photoreagent. Neuraminidase pretreatment of L1210 cells increased mobility of the p118, suggesting that it was glycosylated and, thus, of plasma membrane origin. In transport-deficient L1210 cells, p118 and p50 were found to have a slightly higher molecular mass and were accompanied by a less distinct protein band (approximately 100 kDa). These findings indicate the presence of a polyamine binding protein at the surface of murine and human leukemia cells, which could be directly or indirectly related to the polyamine transport apparatus.

Affinity Labels↗

PubMed1995

Use of 4-fluoro-L-ornithine to monitor metabolic flux through the polyamine biosynthetic pathway.

The mechanistic effectiveness of various polyamine analogs and enzyme inhibitors is typically determined by their ability to deplete intracellular polyamine pools. In this study, we describe an assay that may prove useful in augmenting this relatively static assessment of drug action. The assay relies upon the substitution of 4-fluoro-L-ornithine (Fl-Orn) for ornithine as a polyamine precursor to provide a means to measure metabolic flux through polyamine pools. At concentrations up to 500 microM, the analog did not inhibit the growth of L1210 murine leukemia cells during incubations of up to 72 hr. Using HPLC, the analog was processed metabolically over time to what was deduced to be 2-fluoroputrescine, 6-fluorospermidine and 6-fluorospermine. The relative proportion of fluorinated polyamine analog to the natural polyamine increased with time and Fl-Orn concentration. The sum of the two was found to be nearly identical to the respective polyamine pool of control cells exposed instead to 500 microM ornithine. This indicates that Fl-Orn was recognized and utilized as a precursor at a rate very similar to that of ornithine itself. Using L1210 cells at different stages of cell growth, it was determined that the metabolic flux through the pools, as indicated by the rate of appearance of individual fluorinated polyamine species, reflected the proliferation status of the cells--non-growing cells failed to incorporate the analog. Likewise, in cell types with varying polyamine pool profiles, such as polyamine enzyme overproducers or those with constitutively different spermidine of spermine ratios, the incorporation of the fluorinated analogs into pools was found to be proportional to the size to the natural polyamine pool. In cells treated with inhibitors of S-adenosylmethionine decarboxylase, Fl-Orn incorporation indicated a total blockade of polyamine synthesis at that enzyme site. Overall, the Fl-Orn assay has demonstrated that polyamine pool profiles generally reflect the rate of flux through the pathway in proliferating cells, suggesting that most intracellular polyamines are freely exchangeable with those undergoing metabolic flux.

Adenosylmethionine Decarboxylase↗

PubMed1995

Cationic porphyrin derivatives as inhibitors of polyamine catabolism.

The effects of six cationic porphyrins on several enzymes involved in polyamine biosynthesis and catabolism have been examined. Both spermidine and spermine synthase were unaffected by the porphyrins at up to 2 mM. By contrast, ornithine and S-adenosylmethionine decarboxylase were inhibited by the nickel and cobalt derivatives of meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin (T4MPyP) with IC50 values in the 10-100 microM region. Spermidine/spermine N1-acetyltransferase (SSAT) and polyamine oxidase (PAO) were highly sensitive to the six meso-substituted cationic porphyrins tested, with Ki values as low as 6 nM for SSAT and 85 nM for PAO. These inhibitors may prove useful in defining the structural features of the active site of these enzymes.

Acetyltransferases↗

PubMed1995

Stable amplification of the S-adenosylmethionine decarboxylase gene in Chinese hamster ovary cells.

A Chinese hamster ovary cell subline (CHO/664) > 1000-fold resistant to the S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, CGP-48664 (4-(aminoiminomethyl)-2,3-dihydro-1H-inden-1-one diaminomethylenehydrazone), has been developed and characterized. The cells were also cross-resistant to the highly specific nucleoside analog inhibitor of AdoMetDC, MDL-73811. These unique cells stably overexpress AdoMetDC due to a 10-16-fold amplification of the AdoMetDC gene, which resulted in a similar increase in AdoMetDC transcript levels. In the presence of 100 microM CGP-48664, the CHO/664 cells displayed AdoMetDC activities similar to the parental line. Following removal of the inhibitor, AdoMetDC activity increased steadily over 20 days to 10-12 times that found in parental CHO cells. Decarboxylated (dc) AdoMet pools accumulated rapidly from < 5 pmol/10(6) cells to approximately 1000-1500 pmol/10(6) cells at 3 days due to diffusion away of intracellular inhibitor and to the depletion of putrescine and spermidine as aminopropyl acceptors in dcAdoMet-mediated synthase reactions. Polyamine pools shifted as putrescine, and spermidine pools were processed forward to spermine. During the period from 3 days to 20 days, dcAdoMet pools fell steadily and eventually stabilized at 100-200 pmol/10(6) cells. Providing excess putrescine at this time as an aminopropyl acceptor rapidly lowered dcAdoMet pools and led to a near normalization of polyamine pools, indicating that both dcAdoMet and putrescine are essential in maintaining steady-state polyamine pool profiles. As with cell line variants that overproduce ornithine decarboxylase, polyamine transport was found to be increased in CHO/664 cells due to an apparent inability of the system to down-regulate polyamine transport in response to polyamine excess. Given the unique metabolic disturbances seen in these cells, we anticipate that in addition to providing a useful system for evaluating the specificity of newly developed AdoMetDC inhibitors, they will undoubtedly prove valuable for investigating the various regulatory interrelationships involved in polyamine homeostasis and possibly other aspects of purine metabolism.

Adenosylmethionine Decarboxylase↗

PubMed1995

Preclinical antitumor efficacy of the polyamine analogue N1, N11-diethylnorspermine administered by multiple injection or continuous infusion.

Certain N-alkylated analogues of the natural polyamine spermine have been found to disrupt polyamine pool homeostasis and inhibit tumor cell growth. The most effective of these analogues, N1, N11-diethylnorspermine (DENSPM), apparently depletes intracellular polyamine pools primarily by inducing the polyamine acetylating enzyme spermidine/spermine N1-acetyltransferase, which contributes to polyamine depletion via increased polyamine excretion and catabolism. In this report, the experimental therapeutic efficacy of DENSPM was further examined with the use of other human solid tumor xenografts, including A121 ovarian carcinoma, A549 lung adenocarcinoma, HT29 colon carcinoma, and SH-1 melanoma, and compared with previously obtained findings with MALME-3M and PANUT-3 human melanomas. In vitro studies indicated that the growth sensitivity of most tumor cell lines to DENSPM was similar, with characteristically flat dose-response curves and IC50s ranging between 0.1 and 1 micrometer the only exception was the HT29 colon carcinoma cell line, which had an IC50 of >100 micrometer. For in vivo studies, DENSPM was administered by i.p. injection to female nude athymic mice at 40 and/or 80 mg/kg 3 times a day (every 8 h) for 6 days or by continuous s.c. infusion with the use of Alzet pumps at 120, 240, or 360 mg/kg/day for 4 days. Treatment began after s.c. tumor xenografts had reached 100-200 mm3. The SH-1 melanoma, A549 lung adenocarcinoma, and A121 ovarian carcinoma xenografts responded well to the i.p. administration of analogue with obvious tumor regressions, long-term tumor growth suppressions, and a significant proportion (up to 40%) of apparent cures (i.e., lack of tumor regrowth). However, in similarity to in vitro findings, HT29 colon carcinoma xenografts responded poorly to DENSPM treatment. Massive induction of N1-acetyltransferase activity and extensive depletion of polyamine pools were consistent findings in most tumor types after in vivo or in vitro treatment with DENSPM. The rapidly growing human LOX melanoma xenograft, however, demonstrated poor induction of N1-acetyltransferase activity and the poorest response to DENSPM treatment. In nude athymic mice with MALME-3M melanoma xenografts, constant infusion delivery of DENSPM resulted in prolonged inhibition of tumor growth and long-term tumor regressions comparable to those produced by multiple i.p. injections. On the basis of the unique structure of DENSPM, novel target and mode of intervention, mild host toxicity, and activity against different human solid tumor xenografts, DENSPM is currently being developed as an antitumor agent in humans.

Adenocarcinoma↗

PubMed1994

Collateral sensitivity of human melanoma multidrug-resistant variants to the polyamine analogue, N1,N11-diethylnorspermine.

Certain N-alkylated analogues of the natural polyamine spermine, such as N1,N11-diethylnorspermine (DENSPM), rapidly deplete intracellular polyamine pools by down-regulating the biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and by potently up-regulating the polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase. On the basis of previously reported antitumor activity in human tumor xenograft model systems, DENSPM is currently undergoing Phase I clinical trials against human melanoma and other solid tumors. The antiproliferative activity of this analogue against the multidrug resistance (MDR) phenotype was examined in three MDR sublines of human melanoma RPMI-7932 cells, which were shown to be 2-to 10-fold resistant to classical MDR agents. These MDR lines had been separately derived using different selecting agents (Lemontt et al., Cancer Res., 48: 6344-6353, 1988). Subline functional resistance due to P-glycoprotein was confirmed by decreased retention of rhodamine 123 relative to parent cells as detected by flow cytometry. Although the three sublines were 2- to 10-fold less sensitive than the parent line to classical MDR-type agents, they were found in dose-response studies to be significantly more sensitive to DENSPM than the parent line. In addition, they showed a distinct cytotoxic response after a 48-h treatment with 10 microM DENSPM, which was not apparent in the parent line. Growth sensitivity of the sublines to the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine, or the S-adenosylmethionine decarboxylase inhibitor, CGP-48664, was found to be similar to parent cells. The ratio of the key biosynthetic enzyme activities for ornithine decarboxylase and S-adenosylmethionine decarboxylase was found to be 3.5- to 5-fold higher in all three sublines, due mainly to increases in the former enzyme. This imbalance produced unusually high putrescine pools. Although DENSPM down-regulation of decarboxylase activities and potent up-regulation of spermidine/spermine N1-acetyltransferase activity occurred similarly in both parent and variant lines, polyamine depletion was greater in the variant lines. Collateral sensitivity of the MDR sublines to DENSPM is partially attributable to the finding that analogue (and spermidine) uptake in the sublines was about 2-fold higher (after 2 h) than in the parent cells. The presence of disturbances in polyamine homeostasis and increased sensitivity to DENSPM in three independently selected cell line variants suggests that they may be generally associated with the MDR phenotype in human melanoma and possibly other tumor cells. The collateral sensitivity of human melanoma MDR variants to DENSPM represents a possible therapeutic indication which should be considered during the ongoing clinical evaluation of this drug.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

CGP 48664, a new S-adenosylmethionine decarboxylase inhibitor with broad spectrum antiproliferative and antitumor activity.

Inhibitors of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC), derived from methylglyoxal-bis(guanylhydrazone) (MGBG), have been shown to have significant antitumor activity in several human solid tumor systems (U. Regenass et al., Cancer Res., 52:4712-4718, 1992). From an ongoing effort to synthesize derivatives with increased enzyme specificity and potency and improved antitumor efficacy, we have now identified CGP 48664, a 4-amidinoindan-1-one 2'-amidinohydrazone (J. Stanek et al., J. Med. Chem., 36:2168-2171, 1993). The compound displays potent inhibition of SAMDC (50% inhibitory concentration, 5 nM), modest inhibition of diamine oxidase (50% inhibitory concentration, 4 microM), and no detectable inhibition of ornithine decarboxylase. CGP 48664 inhibits the growth of a panel of human and mouse tumor cell lines, including one which expresses the multidrug resistance phenotype, with 50% inhibitory concentrations ranging between 0.3 and 3 microM. CGP 48664 does not seem to utilize the polyamine transport carrier system since it competes poorly with spermidine for uptake into L1210 cells (Ki 161 microM) and inhibits the growth of polyamine transport-deficient Chinese hamster ovary cells. Relative to MGBG or previously described MGBG analogues, CGP 48664 accumulates to much lower intracellular concentrations. Treatment of the L1210 cell for 48 h with 3 microM CGP 48664 decreases SAMDC activity to < 10% of control and initiates a compensatory 3-fold rise in ornithine decarboxylase. Consistent with SAMDC inhibition, putrescine pools increase 10-fold, whereas spermidine and spermine pools fall to < 10% of control. In contrast to MGBG, CGP 48664 displays attenuated antimitochondrial activity as indicated by a lack of effect on pyruvate oxidation and mitochondrial DNA levels under treatment conditions which inhibit cell proliferation. Specificity of drug action was indicated further by prevention of L1210 cell growth inhibition by exogenous spermidine or spermine. More convincingly, Chinese hamster ovary cells made approximately 1000-fold resistant by chronic exposure to the analogue were found to selectively overexpress SAMDC mRNA due to gene amplification. The new SAMDC inhibitor showed potent antitumor activity against syngeneic tumors (B16 melanoma and Lewis lung carcinoma) and nude mouse human tumor xenografts (T-24 bladder carcinoma, SK MEL-24 melanoma, and MALME-3M melanoma). On the basis of its novel structure, its apparent specificity of action, and its potent antitumor activity, CGP 48664 is the candidate drug for further preclinical development.

Adenosylmethionine Decarboxylase↗

PubMed1993

Cloning and sequence analysis of the gene and cDNA encoding mouse spermidine/spermine N1-acetyltransferase--a gene uniquely regulated by polyamines and their analogs.

The polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase (SSAT), has been implicated as a critical determinant of polyamine pool maintenance. SSAT has recently been shown to be positively regulated in human cell lines by polyamines and their analogs at the level of mRNA accumulation. Mouse LA-4 lung adenoma cells treated with either spermine or the spermine analog, N1,N12-bis(ethyl)spermine, produced a 2.3 and 6.5-fold increase, respectively, in SSAT mRNA. Prior evidence for transcriptional control of the enzyme prompted investigation of SSAT gene structure and its regulatory elements. The mouse SSAT gene was isolated as a 3650 bp EcoRI fragment from a lambda-J1 Mus saxicola genomic library by hybridization with human SSAT cDNA. An additional 431 bp downstream from the 3' EcoRI site were sequenced from a BamHI fragment (total gene sequence, 4066 bp). The gene contains six exons and five introns. Sequence analysis of the 774 bp of the 5' non-coding region revealed the absence of TATAA or CCAAT sequence motifs and the presence of a number of binding motifs in the 5' region of the gene with consensus binding sequences for transcription factors SP1, AP1, E2F, AP2, PEA-3 and others. The deduced amino acid sequence of the coding region differs from that of the human SSAT cDNA by five amino acids. The 527 bp of the 3' non-coding region contains four possible polyadenylation signal sites of which only one displays a typical consensus sequence. A 940 bp SSAT cDNA was isolated from Mus domesticus (BALB-C) liver lambda gt11 cDNA library. It contains a 5' untranslated region 89 bp in length and a 3' untranslated region 376 bp in length. The amino acid sequence deduced from Mus domesticus differs from that of Mus saxicola by one amino acid, from the hamster cDNA, by four amino acids and from the human cDNA by six amino acids. Further elucidation of the structural features of the SSAT gene may reveal how it is positively regulated by polyamines and their analogs.

Acetyltransferases↗

PubMed1993

Polyamine and polyamine analog regulation of spermidine/spermine N1-acetyltransferase in MALME-3M human melanoma cells.

In MALME-3M human melanoma cells the polyamine analog N1,N12-bis(ethyl)spermine (BESPM) suppresses the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, and increases the polyamine catabolizing enzyme, spermidine/spermine N1-acetyl-transferase (SSAT) by more than 200-fold. In the present study increases in SSAT activity in MALME-3M cells treated with 10 microM BESPM were found to be accompanied by a substantial (up to 45-fold) accumulation of SSAT mRNA. By Northern blot analysis three RNA transcripts were found to hybridize with the coding region of human SSAT cDNA: a minor high molecular weight (approximately 3.5 kilobases) species designated form A and two lower molecular weight species designated forms B and C (approximately 1.5 and approximately 1.3 kilobases, respectively). Form A increased uniformly during BESPM treatment and was most obvious in nuclear RNA preparations. On the basis of size similarity to the transcribing region of the gene and hybridization with the coding region of SSAT cDNA and its prevalence in nuclear mRNA preparations, form A is thought to represent precursor SSAT RNA. Form C is present in control cells and increases steadily during treatment, whereas form B increases transiently during early treatment (1-3 h). By RNase H digestion assay, form B was found to have a 200-base pair longer poly(A) tract and as such may represent a precursor to form C. Accumulation of SSAT mRNA was found to be a result of increased gene transcription and stabilization of SSAT mRNA. Nuclear run-on studies indicated a 2-4-fold increase in the transcription rate of the SSAT gene. As indicated by actinomycin D studies, the SSAT mRNA half-life increased with BESPM treatment from 17 to 64 h. The natural polyamine, spermine, also increased SSAT mRNA (5.5-fold at 24 h) and behaved similarly to BESPM in inducing the appearance of the same three transcript forms. The polyamine was much less effective than the analog at increasing enzyme activity. Lowering intracellular polyamine pools with inhibitors of biosynthesis decreased basal SSAT mRNA levels by at least 70% indicating, that the gene can be down-regulated as well as up-regulated by polyamines. These findings indicate that SSAT represents a unique example of gene expression being positively influenced at the RNA level by polyamines and their analogs.

Acetyltransferases↗

PubMed1993

Regulation of spermidine/spermine N1-acetyltransferase by intracellular polyamine pools. Evidence for a functional role in polyamine homeostasis.

Through its role in polyamine acetylation and the back-conversion pathway, spermidine/spermine N1-acetyltransferase (SSAT) has the potential to control intracellular polyamine pools by facilitating their catabolism and/or excretion. The possibility that the enzyme is subject to regulation by intracellular polyamine pools was investigated in MALME-3 human melanoma cells. Increases in intracellular polyamine pools by treatment with 3 microM exogenous spermidine or spermine for 48 h caused SSAT activity to increase 111% and 226%, respectively, and SSAT-specific mRNA to rise 19% and 66%, respectively. Decreases in polyamine pools by treatment with inhibitors of polyamine biosynthesis caused SSAT activity to decrease by 46% and mRNA to fall by 89%. Both SSAT activity and mRNA were more sensitive to changes in spermine than spermidine. The identification of a positive regulatory relationship between SSAT and intracellular polyamine pools further implicates this enzyme in a proposed model for polyamine pool homeostasis.

Acetyltransferases↗

PubMed1993

Antitumor activity of N1,N11-bis(ethyl)norspermine against human melanoma xenografts and possible biochemical correlates of drug action.

In in vitro systems, the spermine analogue, N1,N11-bis(ethyl)norspermine (BENSPM), suppresses the polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase (ornithine decarboxylase and S-adenosylmethionine decarboxylase, respectively), greatly induces the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), depletes polyamine pools, and inhibits cell growth. Against MALME-3 M human melanoma xenografts, BENSPM and related homologues demonstrate potent antitumor activity that has been found to correlate positively with their ability to induce SSAT activity in vitro. Herein, we further evaluate the antitumor activity of BENSPM and at the same time characterize the biochemical effects of BENSPM treatment on polyamine metabolism of selected normal and tumor tissues. At 40 mg/kg 3 times/day for 6 days i.p., BENSPM suppressed growth of MALME-3 M human melanoma xenografts during treatment and for 65 days afterwards. Similar antitumor activity was obtained with 120 mg/kg once daily for 6 days and 40 mg/kg once daily for 6 days, indicating that against this tumor model, the dosing schedule can be relaxed up to sixfold without compromising antitumor activity. When MALME-3 M tumor-bearing mice were retreated with BENSPM 2 weeks after the first treatment at 40 mg/kg 3 times/day for 6 days, initial tumor volumes of 85 mm3 were reduced to < 10 mm3. Analysis of melanoma, liver, and kidney tissues from mice treated with 40 mg/kg 3 times/day for 6 days revealed relatively similar accumulations of BENSPM in all tissues at levels greater than the original total content of polyamine pools. By 2 weeks following treatment, BENSPM pools in normal tissues were almost gone, whereas in tumor tissues significant amounts (40%) were still retained. The biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, gave no indication of enzyme suppression (or increase) by the analogue as typically occurs in vitro. By contrast, SSAT was induced from an average of < 50 pmol/min/mg in control tissues to 320 pmol/min/mg in liver, 1255 pmol/min/mg in kidney, and 13,710 pmol/min/mg in MALME-3M tumor. Two weeks later, SSAT activity was still 12 times higher in tumor than in kidney. Polyamine pools (putrescine, spermidine, and spermine) were reduced after treatment in all tissues and approached near-total depletion in the tumor. Good antitumor activity and even more potent induction of SSAT (i.e., 26,680 pmol/min/mg) was also observed in PANUT-3 human melanoma xenografts. Overall, the findings reveal meaningful antitumor activity by BENSPM against 2 human melanoma xenografts and provide in vivo evidence consistent with SSAT-induced polyamine depletion playing a determining role in at least the initial phase of the antitumor response.

Acetyltransferases↗