The polyamine analog PG11047 potentiates the antitumor activity of cisplatin and bevacizumab in preclinical models of lung and prostate cancer.

PURPOSE: PG11047 is a polyamine analog currently in Phase I trials for advanced cancer as a monotherapy and in combination with a number of approved anti-cancer agents. The use of polyamines as a target for antiproliferative therapy is based on findings that cells synthesize polyamines excessively when induced to grow and that polyamine metabolism is frequently dysregulated in cancer. A selective polyamine transport system provides access for PG11047 into rapidly dividing cells to inhibit polyamine biosynthetic enzymes, to induce the polyamine catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO) which could subsequently induce reactive oxygen species that contribute to tumor cell responses to PG11047, and to function as a polyamine with altered function when it binds to natural polyamine binding sites. The objective of the present study was to assess the antitumor effects of PG11047 alone and in combination with approved anti-cancer agents. METHODS: The antitumor efficacy of PG11047 as a single agent, and in combination with cisplatin and bevacizumab, was tested in models of lung (A549) and prostate (DU-145) cancer, respectively. RESULTS: PG11047 significantly inhibited tumor development in both lung and prostate cancer models when administered as a single agent. In the lung cancer model, PG11047 potentiated the antitumor effect of cisplatin. Although potent activity was observed with PG11047 and bevacizumab when administered as single agents in the prostate cancer model, the combination arm significantly enhanced antitumor activity compared with either agent alone. In all experiments, PG11047 was well tolerated with no adverse effects on bodyweight gain. CONCLUSIONS: The preclinical data support the rationale for the current Phase I trials which are assessing PG11047 as a monotherapy and in combination with a number of approved anti-cancer agents including cisplatin and bevacizumab.

Antizyme and antizyme inhibitor activities influence cellular responses to polyamine analogs.

Close structural analogs of spermidine and spermine, polyamine mimetics, are potential chemotheraputic agents as they depress cellular polyamines required for tumor growth. Specific mimetic analogs stimulate synthesis of the regulatory protein antizyme (AZ), which not only inactivates the initial enzyme in polyamine biosynthesis but also inhibits cellular uptake of polyamines. The role of AZ induction in influencing cellular uptake of representative analogs was investigated using three analogs produced by Cellgate Inc., CGC-11047, CGC-11102, and CGC-11144, which exhibit markedly distinct AZ-inducing potential. An inverse correlation was noted between the AZ-inducing activity of a compound and the steady-state levels accumulated in cells. As some tumor cells over express AZI as a means of enhancing the polyamines required for aggressive growth, analog sensitivity was examined in transgenic CHO cells expressing exogenous antizyme inhibitor protein (AZI). Although AZI over expression increased cell sensitivity to analogs, the degree of this affect varied with the analog used.

Molecular basis of inward rectification: structural features of the blocker defined by extended polyamine analogs.

Polyamines cause inward rectification of Kir K(+) channels by blocking deep within the channel pore. We investigated structural constraints of polyamine block of strongly rectifying mutant K(ATP) channels (Kir6.2[L164C,N160D,C166S] + SUR1). We studied three groups of polyamine analogs: 1) conformationally restricted linear tetra-amines with a cycloalkyl or alkene group between the second and third amines (CGC-11047, CGC-11093, CGC-11099, and CGC-11098), 2) conformationally restricted linear deca-amines with a cycloalkyl or alkene group between the fifth and sixth amines (CGC-11150, CGC-11179, and CGC-11241), and 3) cyclic tetra-amines (CGC-11174, CGC-11197, CGC-11199, and CGC-11254). All linear analogs cause a voltage-dependent block similar to that of spermine, but slightly weaker (at 1 microM, V(1/2) for spermine block = -10 +/- 1 mV, Z = 2.9 +/- 0.1, n = 19; V(1/2) for analogs varies from polyamine -7 to +10 mV, Z = 2.6-3.9). These data indicate tolerance for conformational restriction and an upper limit to the voltage dependence of the blocking process. There was no voltage-dependent block by the cyclic compounds; instead, they induce irreversible rundown of the current. Structural models of Kir channels suggest that a narrow entry at the top of the cytoplasmic pore may exclude cyclic analogs from the inner cavity, thereby explaining the structure-activity relationship that we observe.

Conformationally restricted analogues of 1N,14N-bisethylhomospermine (BE-4-4-4): synthesis and growth inhibitory effects on human prostate cancer cells.

Twelve analogues of 1N,14N-bisethylhomospermine (BE-4-4-4) with restricted conformations were synthesized in the search for cancer chemotherapeutic agents with higher cytotoxic activities and lower systemic toxicities than BE-4-4-4. The central butane segment of BE-4-4-4 was replaced with a 1,2-substituted cyclopropane ring, a 1,2-substituted cyclobutane ring, and a 2-butene residue. In each case, the cis/trans-isomeric pair was synthesized. Cis-monounsaturation(s) was also introduced at the outer butane segment(s) of BE-4-4-4. The two possible cis-dienes and a cis-triene formally derived from the tetraazaeicosane skeleton of BE-4-4-4 were also prepared. Four cultured human prostate cancer cell lines (LnCap, DU145, DuPro, and PC-3) were treated with the new tetramines to examine their effects on cell growth with a MTT assay. One representative cell line (DuPro) was selected to further study the cellular uptake of the novel tetramines, their effects on intracellular polyamine pools, and their cytotoxicity. All tetramines entered the cells, reduced cellular putrescine and spermidine pools while exerting only a small effect on the spermine pool, inhibited cell growth, and killed 2-3 logs of cells after 6 days of treatment at 10 microM. Four new tetramines, the two cyclopropyl isomers, the trans-cyclobutyl isomer, and the (5Z)-tetraazaeicosene, were more cytotoxic than their saturated counterpart (BE-4-4-4). Their cytotoxicity, however, could not be correlated either with their cellular uptake or with their ability to deplete intracellular polyamine pools. We attribute their cytotoxicity to their specific molecular structures. The cytotoxicity was markedly reduced when the central butane segment was deprived of its rotational freedom by replacing it with a double bond. Introduction of a triple bond or a benzene-1,2-dimethyl residue at the central segment of the polyamine chain, led to complete loss of biological activity. The conformationally restricted alicyclic derivatives were not only more cytotoxic than was the freely rotating BE-4-4-4 by several orders of magnitude but also had much lower systemic toxicities than the latter. Thus, we obtained new tetramines with a wider therapeutic window than BE-4-4-4.

cis-Unsaturated analogues of 3,8,13,18,23-pentaazapentacosane (BE-4-4-4-4): synthesis and growth inhibitory effects on human prostate cancer cell lines.

From the results of our previous physicochemical studies of polyamine-nucleic acid interactions, we concluded that polyamine analogues in cisoidal conformation are capable of wrapping around the major groove of the double helix, of displacing natural polyamines from their nucleic acid binding sites, and of inhibiting cell division. On the basis of this hypothesis, nine unsaturated pentamines, formally derived from the cytotoxic pentamine 3,8,13,18,23-pentaazapentacosane (BE-4-4-4-4), were prepared in an attempt to increase antineoplastic activity. Cis-double bonds were introduced in all possible sites in the saturated pentaazapentacosane structure of BE-4-4-4-4 to yield two pentacosenes, four pentacosadienes, two pentacosatrienes, and one pentacosatetraene. Cis-double bonds should also provide good targets for mixed-function oxidases that might eliminate the accumulation of unsaturated pentamines in serum, thereby reducing systemic toxicity in animals. We determined the ability of these new pentamines to inhibit growth in four cultured human prostate cancer cell lines (LnCap, DU145, PC-3, and DuPro) using a MTT assay. LnCap and DU145 cells were very sensitive, PC-3 cells were relatively resistant, and DuPro cells were intermediate in sensitivity to most of these synthetic pentamines. In all cell lines, pentamines that had unsaturation(s) at the end of the chain showed the highest cell growth inhibitory effects. The cellular uptake, effects on cellular polyamine levels, and cytotoxicity of these pentamines on one representative prostate cancer cell line (DuPro) were further examined with a colony-forming efficiency (CFE) assay. The pentamines with unsaturation(s) at the end of the chain were once again the most cytotoxic among both the saturated (BE-4-4-4-4) and unsaturated analogues. Appreciable amounts of all pentamines entered DuPro cells and depleted cellular polyamine pools by day 6 of treatment. For most pentamines, however, cell growth inhibitory and cytotoxic effects could not be directly correlated either with their cellular uptake or with their ability to deplete cellular polyamine pools. The position of the double bonds in the aliphatic backbone seems to be the most important determinant of cytotoxicity. For some pentamines, however, depletion of cellular polyamines may add to their efficacy.

[(1)N,(12)N]Bis(Ethyl)-cis-6,7-dehydrospermine: a new drug for treatment and prevention of Cryptosporidium parvum infection of mice deficient in T-cell receptor alpha.

Cryptosporidium parvum infection of T-cell receptor alpha (TCR-alpha)-deficient mice results in a persistent infection. In this study, treatment with a polyamine analogue (SL-11047) prevented C. parvum infection in suckling TCR-alpha-deficient mice and cleared an existing infection in older mice. Treatment with putrescine, while capable of preventing infection, did not clear C. parvum from previously infected mice. These findings provide further evidence that polyamine metabolic pathways are targets for new anticryptosporidial chemotherapeutic agents.

Polyamines decrease Ca(2+) sensitivity of tension and increase rates of activation in skinned cardiac myocytes.

Owing in part to their interactions with membrane proteins, polyamines (e.g., spermine, spermidine, and putrescine) have been identified as potential modulators of membrane excitability and Ca(2+) homeostasis in cardiac myocytes. To investigate whether polyamines also affect cardiac myofilament proteins, we assessed the effects of polyamines on contractility using rat myocytes and trabeculae that had been permeabilized with Triton X-100. Spermine, spermidine, and putrescine reversibly increased the [Ca(2+)] required for half-maximal tension (i.e., right-shifted tension pCa curves), with the following order of efficacy: spermine (+4) > spermidine (+3) > putrescine (+2). However, synthetic analogs that differed from spermine in charge distribution were not as effective as spermine in altering isometric tension. None of the polyamines had a significant effect on maximal tension, except at high concentrations. After flash photolysis of DM-Nitrophen (a caged Ca(2+) chelator), spermine accelerated the rate of tension development at low and intermediate but not high [Ca(2+)]. These results indicate that polyamines, especially spermine, interact with myofilament proteins to reduce apparent Ca(2+) binding affinity and speed cross-bridge cycling kinetics at submaximal [Ca(2+)].

Conformationally restricted analogues of 1N,12N-bisethylspermine: synthesis and growth inhibitory effects on human tumor cell lines.

Eight analogues of 1N,12N-bisethylspermine (BES) with restricted conformations were synthesized in the search for new spermine mimetics with cytotoxic activities. By replacing the central butane segment of BES with a 1,2-disubstituted cyclopropane ring, a pair of cis/trans-isomers was obtained that introduced a spatial constraint in the otherwise freely mobile butane chain. An analogous pair of isomers was obtained when the butane segment was replaced with a 1, 2-disubstituted cyclobutane ring or with a 2-butene residue. The six new BES analogues thus obtained (three pairs of cis/trans-isomers) were growth inhibitory at low-micromolar concentrations against four human tumor cell lines (A549, HT-29, U251MG, and DU145) but were less growth inhibitory against two other human tumor cell lines (PC-3 and MCF7). 1N,12N-Bisethylspermyne, where the central butane segment of BES was replaced by the rigid 2-butyne segment, was devoid of growth inhibitory activity against five of the six human cell lines studied (DU145 being the only exception), a clear indication of the importance of conformational mobility at the 4N, 9N-butane segment of BES for its biological activity. When the butane segment was replaced by a benzene-1,2-dimethyl residue, the resulting BES analogue was devoid of growth inhibitory activity despite its cisoid conformation. The cytotoxicity of the analogues does not seem to be directly related to their uptake by the cells or to their effects on cellular polyamine levels. BES analogues with restricted conformations but which contained the equivalent of a two-carbon unit, rather than the natural four-carbon unit, at the central segment, such as 1,2-diaminocyclopropyl or 1, 2-diaminocyclobutyl derivatives, were devoid of growth inhibitory effects at the concentrations studied. The development of conformationally restricted polyamine analogues appears to show promise in the further quest for polyamine-related therapeutic agents with specificity of action.

Effects of 1,2-naphthoquinones on human tumor cell growth and lack of cross-resistance with other anticancer agents.

The sensitivity of human tumor and rat prostate tumor cells to a series of naphthoquinones, including tricyclic compounds of the beta-lapachone and dunnione families as well as 4-alkoxy-1,2-naphthoquinones, was evaluated. To better understand the mechanism of cytotoxicity of 1,2-naphthoquinones, the roles of various resistance mechanisms including P-glycoprotein, multidrug resistant associated protein, glutathione (GSH) and related enzymes, altered topoisomerase activity, and overexpression of genes that control apoptosis (bcl-2 and bc-xL) were studied. MCF7 cells were most sensitive to the naphthoquinones with IC50 values ranging from 1.1 to 10.8 microM, as compared to 2.5 to >32 microM for HT29 human colon, A549 human lung, CEM leukemia and AT3.1 rat prostate cancer cells. MCF7 ADR cells, selected for resistance to adriamycin (ADR), displayed cross-resistance to the tricyclic 1,2-naphthoquinones. Drug efflux via a P-glycoprotein mechanism was ruled out as a mechanism of resistance to 1,2-naphthoquinones, since KB-V1 cells expressing high levels of P-glycoprotein and the KB-3.1 parent line were equally sensitive to these compounds. Any resistance of the tricyclic naphthoquinones noted in ADR-resistant cells appeared to relate to the GSH redox cycle and could be circumvented by exposure to buthionine sulfoximine or by changing the structure from a tricyclic derivative to a 4-alkoxy-1,2-naphthoquinone. The 1,2-naphthoquinones were found to be cytotoxic against CEM/VM-1 and CEM/M70-B1 cells that were selected for resistance to teniposide or merbarone, respectively. In addition, cells overexpressing bcl-2 or bcl-xL proteins were as sensitive to 1,2-naphthoquinones as were control cells. Because of their effectiveness in drug-resistant cells, these agents appear to hold promise as effective chemotherapeutic agents.

Reaction of beta-lapachone and related naphthoquinones with 2-mercaptoethanol: a biomimetic model of topoisomerase II poisoning by quinones.

1,2-Naphthoquinones, such as beta-lapachone, 4-alkoxy-1,2-naphthoquinones, and tetrahydrofuran-1,2-naphthoquinones, react rapidly with 2-mercaptoethanol in benzene to give 1,4-, 1,2-, 1,3- and 1,6-Michael-type adducts that are formed by the addition of the thiol group to the quinone ring. Menadione (2-methyl-1,4-naphthoquinone) reacts with the thiol reagent very slowly under the same reaction conditions. Although the formation of the adducts can be followed by 1H-NMR, attempts to isolate the adducts failed due to their retroconversion to the starting products. On addition of a Lewis acid, however, the adducts undergo cyclization reactions that give stable derivatives that can be isolated and characterized. Determination of the structures of the derivatives allowed for the identification of the adducts from which they originated. Thus, beta-lapachone and 2,3-dinordunnione underwent 1,4- and 1,2-Michael type additions to the quinone ring, while 4-pentyloxy-1,2-naphthoquinone underwent two simultaneous Michael additions to the quinone ring of the naphthoquinone. Menadione underwent a single 1,3-addition. The alkylation rates of the thiol group of 2-mercaptoethanol by the naphthoquinones parallel the naphthoquinones efficiencies in inducing DNA cleavage through DNA-bound topoisomerase II. These results support our hypothesis that the cytotoxic effect of the naphthoquinones derive, at least in part, from their alkylation of exposed thiol residues on the topoisomerase II-DNA complex.

Effects of polyamine analogues on prostatic adenocarcinoma cells in vitro and in vivo.

PURPOSE: The overall purpose of this study was to determine the potential usefulness of 1,19-di-(ethylamino)-5,10,15-triazononadecane (BE-4-4-4-4) in the treatment of prostate cancer using in vitro and in vivo models. More specifically the objectives were: (1) to determine the in vitro and in vivo sensitivity of human and rat prostate cancer cells to two polyamine analogues N1,N11-di(ethyl)norspermine (DENSPM) and BE-4-4-4-4; (2) to determine whether the mechanism of cell kill occurred through an apoptotic pathway; and (3) to determine the toxicity associated with therapeutic doses of BE-4-4-4-4 using an animal model. METHODS: In order to determine the ability of these drugs to cause in vitro cytotoxicity, colony-forming assays were performed utilizing the well-characterized Dunning rat prostate cancer cell lines AT3.1, AT6.1 and AT6.3, and the androgen-insensitive human prostate cancer cell lines DU145, DuPro-1 and TSU-Pr1. Apoptotic cell death was determined using DNA laddering and DAPI staining of nuclei. The antitumor activity of BE-4-4-4-4 was evaluated by treatment of DuPro- and PC-3 xenograft tumors in nude mice. RESULTS: BE-4-4-4-4 was shown to be approximately 4 to 86 times more cytotoxic in clonogenic assays than DENSPM in both rat and human prostate carcinoma cell lines. Cells treated with cytotoxic doses of DENSPM or BE-4-4-4-4 showed no signs of apoptosis using either DNA laddering or DAPI staining of nuclei. There was a significant inhibition of DuPro-1 tumors for animals treated with BE-4-4-4-4 compared with control animals. Equitoxic doses of BE-4-4-4-4 resulted in greater tumor inhibition than DENSPM, although the difference was not significant. After treatment with therapeutic doses of BE-4-4-4-4, histopathologic evaluation indicated minimal to mild necrosis and inflammation in the kidneys on days 15 and 22 following treatment. On day 35, there was no necrosis or regeneration present in the kidney, indicating that the toxicity was transient and that regeneration of epithelial cells was complete with apparent return to normalcy. CONCLUSIONS: These initial studies demonstrate that BE-4-4-4-4 is cytotoxic against rat and human prostate cancer cells in culture and effective against DuPro-1 xenografts in nude mice. Polyamine analogues, such as DENSPM or BE-4-4-4-4, should be considered for clinical use in the treatment of prostate adenocarcinomas.

Somatic mutational mechanisms involved in intestinal tumor formation in Min mice.

We have demonstrated previously that intestinal tumor formation in B6 Min/+ mice is always accompanied by loss of the wild-type adenomatous polyoposis coli (Apc) allele and that intestinal tumor multiplicity in B6 Min/+ mice can be significantly increased by treatment with a single dose of N-ethyl-N-nitrosourea (ENU). Here, we show that some tumors from ENU-treated Min/+ mice can form without complete elimination of Apc+. At least 25% of these tumors acquired somatic Apc truncation mutations. Interestingly, some ENU-induced tumors demonstrated loss of the Apc+ allelic marker examined by the quantitative PCR assay used here. Using two methods of mutation detection, we identified no Apc mutations in at least 12% of the tumors from ENU-treated B6 Min/+ mice. Finally, no H- or K-ras-activating mutations were detected in intestinal tumors from either untreated or ENU-treated Min/+ mice. The majority of somatic human APC mutations in intestinal tumors lead to APC truncation. Our results demonstrate that somatic Apc truncation mutations also frequently occur in ENU-induced intestinal tumors in Min mice.

Induction of DNA topoisomerase II-mediated DNA cleavage by beta-lapachone and related naphthoquinones.

Recent studies have suggested that 3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione (beta-lapachone) inhibits DNA topoisomerase I by a mechanism distinct from that of camptothecin. To study the mechanism of action of beta-lapachone, a series of beta-lapachone and related naphthoquinones were synthesized, and their activity against drug-sensitive and -resistant cell lines and purified human DNA topoisomerases as evaluated. Consistent with the previous report, beta-lapachone does not induce topoisomerase I-mediated DNA breaks. However, beta-lapachone and related naphthoquinones, like menadione, induce protein-linked DNA breaks in the presence of purified human DNA topoisomerase IIalpha. Poisoning of topoisomerase IIalpha by beta-lapachone and related naphthoquinones is independent of ATP and involves the formation of reversible cleavable complexes. The structural similarity between menadione, a para-quinone, and beta-lapachone, an ortho-quinone, together with their similar activity in poisoning topoisomerase IIalpha, suggests a common mechanism of action involving chemical reactivity of these quinones. Indeed, both quinones form adducts with mercaptoethanol, and beta-lapachone is 10-fold more reactive. There is an apparent correlation between the rates of the adduct formation with thiols and of the topoisomerase II-poisoning activity of the aforementioned quinones. In preliminary studies, beta-lapachone and related naphthoquinones are found to be cytotoxic against a panel of drug-sensitive and drug-resistant tumor cell lines, including MDR1-overexpressing cell lines, camptothecin-resistant cell lines, and the atypical multidrug-resistant CEM/V-1 cell line.

Capturing the promise of science in medical schools.

To gain a better understanding of the effects on medical schools of transformations in medical practice, science, and public expectations, the Association of American Medical Colleges (AAMC) constituted the Advisory Panel on the Mission and Organization of Medical Schools (APMOMS) in 1994. APMOMS created six working groups to address the issues deemed by panel members to be of highest priority. This article is a report of the findings of the Working Group on Capturing the Promise of Medical Research, which addressed questions concerning the direction of research and the integration of scientific developments in medical education and practice. The working group explored a broad panorama of issues, including those related to sustaining the accomplishments, momentum, and progress of medical research. A dominant theme emerged: the central importance of an environment of discovery to the core missions of medical schools. The present article consists of the group's comments and recommendations on the main topic-the promise of biomedical research in relation to medical education-and their comments and recommendations on five other topics that have important relationships to the main topic and to the group's central charge. These are ethics; academia-industry relations; the administrative structure of medical schools; university-medical school relations; and research funding.

Preserving medical schools' academic mission in a competitive marketplace.

To gain a better understanding of the effects on medical schools of transformations in medical practice, science, and public expectations, the AAMC in 1994 formed the Advisory Panel on the Mission and Organization of Medical Schools and appointed six working groups to address relevant issues. This article is a report of the findings of the Working Group on Preserving Medical Schools' Academic Mission in a Competitive Marketplace, which was charged with exploring how medical schools could acquire and/or preserve an adequate patient base for teaching, research, and income generation in a competitive marketplace. The other groups' reports will appear in future issues of Academic Medicine. To understand the diversity of approaches that schools have taken to achieve this goal and to preserve their missions, the group interviewed representatives of nine medical schools, selected to represent a cross section of U.S. medical schools. The interviews took place on four occasions between June 1995 and March 1996. The information and comments shared by participants helped the working group gain insight into the fundamental issues it had been charged to address, including those of new delivery structures, what value schools offer to delivery structures, how education and research can be incorporated and supported financially, possible new pressures on relationships between medical schools and teaching hospitals, changes in faculty physicians' employment relationships and terms, and the role of the medical school in graduate medical education. In collecting and analyzing the data, the working group focused on the distinction between protecting an institution's existing enterprise and preserving an institution's core mission. This article gives a detailed overview of the information and comments each school presented, organized under the appropriate question. The working group's conclusions and commentaries on the findings follow. An appendix presents more detailed summaries of the schools' presentations, organised as case studies. The picture that emerges is complex. The working group concluded that medical schools will take a variety of approaches to define and preserve their missions. Most, but not all, medical schools will be able to secure the patient bases necessary to fulfill their missions even in a competitive marketplace. However, the nature of many of the schools is likely to change, and it is not clear whether the core missions of education and research will continue at their present levels at all schools.

The ability of polyamine analogues to induce Z-DNA structure in synthetic polynucleotides in vitro inversely correlates with their effects on cytotoxicity of cis-diaminedichloroplatinum (II) (CDDP) in human brain tumor cell lines.

We studied the effects of 72 h pretreatment with five polyamine analogues on the cytotoxicity of cis-diaminedichloroplatinum (II) (CDDP) in U-251 MG and SF-188 human brain tumor cells. A colony forming efficiency assay showed that the pretreatment with clinically important analogues 1,11-bis(ethylamino)-4, 8-diazaundecane (BE-3-3-3), 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4), and 1,l9-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) increased the cytotoxicity of CDDP by 1.3 to 2.3-fold; 1,19-diamino-5,10, 15-triazanonadecane (4-4-4-4) did not affect CDDP cytotoxicity, and 1,11-diamino-4,8-diazaundecane (3-3-3) protected cells from the cytotoxic effects of CDDP. An alkaline elution assay detected a small increase in DNA interstrand crosslinks accompanying the enhancement of CDDP cytotoxicity only in cells pretreated with BE-3-3-3. This study is the first to show that the Z-DNA inducing abilities of the polyamine analogues in synthetic polynucleotides in vitro correlates inversely with their effects on CDDP cytotoxicity in human tumor cells in culture.

A new model for disruption of the ornithine decarboxylase gene, SPE1, in Saccharomyces cerevisiae exhibits growth arrest and genetic instability at the MAT locus.

Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway. We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines. Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene. Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1. Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen. All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation. A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination.

Spermine induces haemoglobin synthesis in murine erythroleukaemia cells.

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. Haemoglobin production was accompanied by accumulation of cytoplasmic beta-globin mRNA and growth inhibition, but not by cell-cycle block or changes in cell volume. Hexamethylene-bisacetamide (HMBA), a well known differentiating agent, also induces haemoglobin production, but causes a G1 block and decreases cell volume. These findings indicate that HMBA and spermine affect MEL cells differently, even though both induce haemoglobin production.

The structure of polyamine analogues determines haemoglobin production and cytotoxicity in murine erythroleukaemia cells.

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. We have studied the ability of various polyamine analogues to inhibit cell growth and induce haemoglobin production. Polyamine analogues with free terminal amino groups were good inducers of haemoglobin production in MEL cells. Haemoglobin levels correlated with the number of positive charges: pentamines (five positive charges) were stronger inducers than tetramines (four positive charges). Compounds ethylated at their terminal amines were poor inducers of haemoglobin production but good inhibitors of MEL cell growth. These results provide evidence that polyamine analogues support specific biological functions of polyamines in MEL cells and suggest relationships between polyamine structure and function.