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Pentamidine and berenil, clinical antiparasitic amidines, have been found to be potent competitive inhibitors of human spermidine/spermine acetyltransferase (SSAT). Ki values were found to be 2.4 and 2 microM, respectively, with spermidine as substrate. A second enzyme of polyamine back-conversion, murine polyamine oxidase (PAO), was found to be competitively inhibited by pentamidine, with a Ki of 7.6 microM when N-acetylspermine was the substrate. Berenil, on the other hand, was an extremely weak inhibitor (Ki = 120 microM). The implication of the effect of inhibition of polyamine back-conversion on the growth of mammalian parasites is discussed.

Trichomonas vaginalis grown for 16 h in the presence of [(14)C]spermine formed a high intracellular pool of [(14)C]spermidine and a small but detectable pool of [(14)C]putrescine. When [(3)H]putrescine was added to the growth medium, a large intracellular pool of [(3)H]putrescine was found, but it was not further metabolized, confirming previous studies suggesting the absence of a forward-directed polyamine synthetic pathway in T. vaginalis. Spermidine:spermineN:(1)-acetyltransferase (SSAT) and polyamine oxidase enzyme activities were detected which collectively converted spermine to spermidine. Polyamine oxidase was localized in the hydrogenosome-enriched fraction, whereas SSAT was found predominantly in the cytosolic fraction. In the presence of saturating substrate, the trichomonad SSAT had an activity of 0. 39+/-0.09 nmol min(-1) (mg protein)(-1) (the mean of five analyses) and an apparent K:(m) for spermine of 1.7 microM. The enzyme was competitively inhibited by di(ethyl)norspermine with a K:(i) of 28 microM. Growth studies indicated that 50 microM di(ethyl)norspermine caused a 68% and 84% reduction in the intracellular concentrations of spermidine and spermine, respectively. The trichomonad polyamine oxidase required FAD as a cofactor and had an apparent K:(m) of 6.0 microM for N(1)-acetylspermine. The potential of bis(alkyl) polyamine analogues as antitrichomonad agents is discussed.

Acetylation of polyamines by spermidine/spermine N(1)-acetyltransferase (SSAT) has been implicated in their degradation and/or export out of the cell. The relationship of SSAT to polyamine pool dynamics and cell growth is not yet clearly understood. MCF-7 human breast carcinoma cells were transfected with tetracycline-regulated (Tet-off) SSAT human cDNA or murine gene. Doxycycline removal for >2 days caused a approximately 20-fold increase in SSAT RNA and a approximately 10-fold increase in enzyme activity. After 4 days, intracellular putrescine and spermidine pools were markedly lowered, and cell growth was inhibited. Growth inhibition could not be prevented with exogenous polyamines due to a previously unrecognized ability of SSAT to rapidly acetylate influxing polyamines and thereby prevent restoration of the endogenous pools. Instead, cells accumulated high levels of N(1)-acetylspermidine, N(1)-acetylspermine, and N(1), N(12)-diacetylspermine, a metabolite not previously reported in mammalian cells. Doxycycline deprivation before treatment with N(1), N(11)-diethylnorspermine markedly increased analog induction of SSAT mRNA and activity and enhanced growth sensitivity to the analog by approximately 100-fold. Overall, the findings demonstrate that conditional overexpression of SSAT lowers polyamine pools, inhibits cell growth, and markedly enhances growth sensitivity to certain analogs. The enzyme also plays a remarkably efficient role in maintaining polyamine pool homeostasis during challenges with exogenous polyamines.

The binding of spermine (SPM), N¹,N12-bismethylspermine (BMS) and N¹,N12-bisethylspermine (BES) to tRNA Phe was studied using ¹H-NMR at 750 MHz. The polyamines were enriched in 13C at the 5-CH2 and 8-CH2 residues and the nuclear Overhauser enhancement (NOE) cross peaks connecting the ¹H-NMR resonances of the13C-methylenes and several base paired imino protons of tRNA Phe were obtained using 1D13C-half filteredspectra. It was found that while SPM and BMS bind to the N(3)-H of base pairs T54-m¹A58, U50-A64 and U52-A62, BES binds only to T54-m¹A58 and U50-A64. This regioselectivity in the binding of the three polyamines to tRNA was correlated with their biological effects on cell growth. Using human melanoma cancer cells (MALME-3M), we found that SPM and BMS were without effect and cytostatic, respectively, while BES was distinctly cytotoxic. The latter also affected cell cycling and, at variance with SPM and BMS, lead to a distinctG1/S cell cycle arrest. Neste trabalho foi feito o estudo de ligação de espermina (SPM), N¹, N12- bismetilespermina (BMS) e N¹, N12-bisetilespermina (BES) ao tRNAfenusando RMN de¹H a 750 MHz. As poliaminas foram enriquecidas com13C nos resíduos 5-CH2 e 8-CH2, sendo obtidos os picos cruzados por efeito de Overhauser nuclear entre as ressonâncias dos hidrogênios dos metilenos marcados com 13C e vários hidrogênios de grupos imino de pares debases do tRNAfen através de espectros 1D filtrados por13C. Foi encontrado que, enquanto SPM e BMS se ligam ao N(3)-H dos pares de bases T54-m¹A58, U50-A64 e U52-A62, BES liga-se só nos pares T54-m¹A58 e U50-A64. Esta regiosseletividade de ligação das três poliaminas ao tRNA foicorrelacionada com seus efeitos biológicos no crescimento celular. Usando células de câncer de melanoma humano (MALME-3M), foi encontrado que SPM e BMS não tem efeito e é citostático, respectivamente, enquanto que BES é claramente citotóxica. Esta última poliamina também afeta o ciclo celular e, contrário aocomportamento de SPM e BMS, acarreta a uma clara parada do ciclo celular G1 /S.

A systematic investigation of the impact of spermidine analogues both in vitro and in vivo is described. The study characterizes the effects of these analogues on L1210 cell growth, polyamine pools, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, spermidine/spermine N1-acetyltransferase, the maintenance of cellular charge, i.e., cationic equivalence associated with the polyamines and their analogues, and compares their ability to compete with spermidine for transport. The findings clearly demonstrate that the activity of the linear polyamine analogues is highly dependent on the length of the triamines and the size of the N(alpha),N(omega)-substituents. It appears that there is an optimum chain length for various activities and that the larger the N(alpha),N(omega)-alkyls, the less active the compound. Metabolic transformation including N-dealkylation of these compounds is also evaluated. While there is no monotonic relationship between chain length and the ability of the analogue to be metabolized, the dipropyl triamines are clearly more actively catabolized than the corresponding methyl and ethyl systems. A comparison of the triamines with the corresponding tetraamines is made throughout the text regarding both in vitro activity against L1210 cells and in vivo toxicity measurements, suggesting that several triamine analogues may offer therapeutic advantages over the corresponding tetraamines.

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 the6-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.

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.

A series of analogues and homologues of N 1 ,N 12 -diethylspermine (DESPM) was synthesized, and their biological properties were evaluated. These tetraamines include a simple linear analogue of DESPM, N 1 ,N 12 -bis(2,2,2-trifluoroethyl)spermine (FDESPM), the cyclic analogues of DESPM, N,N'-bis(4-piperidinylmethyl)-1,4-diaminobutane [PIP(4,4,4)] and N,N'-bis[2-(4-piperidinyl)ethyl]-1,4-diaminobutane [PIP(5,4,5)], and their aromatic counterparts, N,N'-bis-(4-pyridylmethyl)-1,4-diaminobutane [PYR(4,4,4)] and N,N'-bis[2-(4-pyridyl)ethyl]-1,4-diaminobutane [PYR(5,4,5)]. The analogues FDESPM, PIP(4,4,4), and PYR(4,4,4) have distances between their nitrogen atoms almost identical to those of DESPM. The longer analogues PIP-(5,4,5) and PYR(5,4,5) are very similar in the spacing of their amino groups. However, the pK a of the nitrogens in the groups differ ; thus, the extent of protonation and the charge characteristics among the members of the groups differ. A comparison of the biological properties of these compounds clearly demonstrates that the tetraamines must be charged to be recognized by the cell. Analogues with low nitrogen pK a 's such that the nitrogens are poorly protonated at physiological pH do not compete well with spermidine for uptake and, as expected, have high 96 h IC 50 values and have little effect on S-adenosylmethionine decarboxylase, ornithine decarboxylase, and spermidine/spermine N 1 -acetyltransferase activities and on intracellular polyamine pools

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.

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 λ-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 λgt11 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.

The biochemical and antiproliferative effects of two recently developed N-alkylated analogues of naturally-occurring polyamines, N 1 ,N 14 -diethylhomospermine (DEHSPM) and N 1 ,N 11 -diethylnorspermine (DENSPM), were investigated in two human transitional cell carcinoma (TCC) lines, T24 and J82. Parallel studies with the ornithine decarboxylase enzyme inhibitor α-difluoromethylornithine (DFMO) were included for comparison. DENSPM displayed greater antiproliferative activity than DEHSPM in both TCC cell lines. Both analogues were strikingly more potent than DFMO. DEHSPM and DENSPM suppressed the activity of the major biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase. However, differences in the resulting polyamine depletion suggest that the substantial antiproliferative activity of these analogues may result from mechanisms other than polyamine depletion. The greater polyamine depletion seen with DENSPM is thought to result from its striking induction of spermidine/spermine N 1 -acetyltransferase. DENSPM is an attractive agent for further preclinical and clinical development, possibly as a chemopreventive agent, in TCC of the bladder.

Induction of polyamine biosynthetic activity and the subsequent increases in intracellular polyamine pools are well documented components of the proliferative response (reviewed in 1–3). Indeed, several lines of evidence clearly indicate that sustained polyamine biosynthesis is a critical component of cell growth and not simply a consequence of it (2,4). In many ways, the association of polyamines with cell growth and, in particular, the properties of the enzyme proteins themselves bear intriguing resemblance to proto-oncogenes and their encoded products. Two key biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase (ODC and AdoMetDC, respectively), are extremely short-lived with half-lives of less than 1 hr, highly inducible and subject to sensitive regulatory control. Increases in their activities are invariably associated with the very early stages of cell growth and, somewhat less consistently, with tumor promotion (5,6). Although the most illustrative of the enzymes in this respect, ODC, has not been shown to have transforming capabilities, cells which overexpress the enzyme can be endowed with increased proliferative potential (7) and/or tissue invasiveness (8). Moreover, induction of the enzyme, like certain of the proto-oncogenes, is known to be critically important for initiating and sustaining cell proliferation.

Adenocarcinoma of the pancreas presents a formidable challenge both experimentally and clinically, whereby effective anticancer therapy is lacking. We have recently explored a relatively new class of antitumor agents in pancreatic cancer cell lines and have found the bis-ethyl derivatives of spermine to show considerable promise. In the present paper, we report the results of in vivo studies demonstrating the antitumor activity of two of these N-alkylated analogues, N1,N14-bis(ethyl)homospermine (BEHSPM) and N1,N11-bis(ethyl)norspermine (BENSPM) in athymic (nude) mouse xenografts of two human pancreatic ductal adenocarcinoma cell lines, PANC-1 (poorly differentiated) and BxPC-3 (moderately well-differentiated). BENSPM was found to exert greater antitumor activity in vivo than either BEHSPM or other conventional agents, largely because higher doses could be given due to its lower toxicity to mice. BENSPM shows greater activity than any other agent we have thus far tested against our pancreatic-cancer models. Optimal schedules of administration have yet to be determined. Nevertheless, of the analogues tested, BENSPM presently appears to be the analogue of choice for further development.

N-Alkylated polyamine analogues have been shown to exert antiproliferative effects in several tumor models, with the bis-ethyl derivatives exerting the greatest suppression of polyamines by virtue of down-regulation of the polyamine biosynthetic enzymes. Pancreatic adenocarcinoma presents a challenge both clinically and experimentally due to its inherent resistance to conventional therapy, which results in its having the worst 5-year survival rate of all cancers. We have previously shown that N1,N12-bis(ethyl)spermine (BESPM) is much more potent than the polyamine enzyme inhibitor alpha-difluoromethylornithine (DFMO) against pancreatic adenocarcinoma cell lines. In the present study, we compared the biochemical and antiproliferative effects of two N-alkylated polyamine analogues, N1,N14-bis(ethyl)homospermine (BEHSPM) and N1,N11-bis(ethyl)norspermine (BENSPM) in two human pancreatic ductal adenocarcinoma cell lines, PANC-1 (poorly differentiated) and BxPC-3 (moderately well-differentiated), and in the WD PaCa (well-differentiated ductal) hamster cell line. BENSPM displayed greater antiproliferative activity in the human pancreatic cancer cell lines, whereas BEHSPM was more potent in the hamster cell line. Both BEHSPM and BENSPM suppress the activity of the major biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase. However, the induction of polyamine depletion in the human cell lines was only modest for BENSPM and minimal for BEHSPM, which suggests that the substantial antiproliferative activity of these analogues may result from mechanisms other than polyamine depletion. The somewhat greater polyamine depletion seen following treatment with BENSPM is thought to result from its striking induction of spermidine/spermine N1-acetyltransferase. The biochemical and antiproliferative activity of BENSPM makes it an attractive agent for further preclinical and clinical development, especially in pancreatic cancer.

Introduction: Bortezomib (B) is the first in class proteasome inhibitor that received FDA approval for the treatment of MM patients with relapsed or refractory disease. Despite impressive clinical activity all patients develop resistance to B. The underlying mechanism of resistance to B remains undetermined. Mechanisms to overcome B resistance or development of new therapeutic agent(s) with activity in context of B resistance are limited due to unavailability of established preclinical B resistant MM models. To overcome this challenge we developed a B resistant HMCL. Methods: we chronically exposed the OPM-2 HMCL to sub-lethal doses of B. Surviving cells were harvested, re-cultured and dose of B incrementally increased over prolong period of time. The resulting resistant cells were further characterized using array Comparative Genomic Hybridization (aCGH), spectral karyotyping (SKY) and gene expression profiling. Results: After several passages we were able to induce B resistance in this HMCL. Final clone (OPM-2BR) demonstrate resistance to 2 × IC50 dose of B. While SKY and aCGH analysis demonstrated significant differences when compared with parent OPM-2WT (wild type) HMCL, gene expression profiling of the resistant and parental lines demonstrated significant upregulation in the expression of a number of ATP-binding cassette transporters. For example, the breast cancer resistance protein (ABCG2) is upregulated 4-fold in the resistant cell line compared to the parental cell line. This data suggests that drug efflux mediated by drug transporters represents one potential mechanism of resistance to B. Conclusion: Chronic in vitro B exposure results in induced resistance in HMCL-OPM-2BR. Resistant cell line demonstrates cytogenetic variability when compared to the parent cell line. Induction of resistance is stable and provides an important preclinical tool to investigate mechanism(s) of B resistance as well as new drug development for B resistant myeloma patients. Detailed analysis of this cell lines including therapeutic interventions investigating resistance reversal will be presented at the meeting.

Introduction: In a phase II clinical trial we reported the antileukemic effect of L in CLL pts. In this clinical trial 24/34 (70.5%) pts with detectable tumor cells (ALC > 5,000) in peripheral blood demonstrated a decrease in ALC within 8 days of treatment. Although, the exact molecular mechanism for its antitumor activity remains undetermined, L has been reported to down regulates production of various cytokines including VEGF, IL-6 and TNF-alpha. In order to investigate the underlying mechanism(s) responsible for the antileukemic effects of L in CLL, we examined down stream targets of VEGF and IL-6 signaling pathways in CLL cells obtained from pts pre and post 8 day treatment with L. Method: Pts with accessible tumor cells in blood (ALC > 5000) who received L were identified. Tumor cells were obtained prior to (baseline) and 8 days after treatment with L. Whole cell lysate was prepared and expression of down stream targets of VEGF, AKT and Erk determined through immunobloting assay. Results: In cells obtained from pts who had received 8 days of treatment, we observed that L decreased the expression of pAKT and pErk1/2 without changes in corresponding total protein. Conversely L did not have any effect on protein expression of the Bcl-2 or Bcl-xl, though Mcl-1 was decreased compared to pretreatment control. Conclusion: This is the first time that the antileukemic effect of L in CLL may at least be attributed to down regulation of VEGF and IL-6 mediated prosurvival pathways. Our observation that L does not interrupt the Bcl-2 pathway are clinically intriguing and propose the possibility of combining L with Bcl-2 inhibiting agent(s) to augment the antitumor response in CLL.

Introduction: L is a more potent analogue of thalidomide with antitumor activity reported in MDS and multiple myeloma. Clinical anti-leukemic activity of L is reported for the first time by our group in pts with CLL. The underlying mechanism of its antitumor activity remains undetermined. We investigated the effect of L on the tumor microenvironment and studied the modulation of soluble cytokines and immune cells (T and NK cells) in pts receiving L. Patients and Methods: CLL pts enrolled on the clinical study with L were eligible. Pre and post (day 7) samples were obtained for evaluation of changes in serum cytokine and immune cell environment. Malignant cells were also obtained to investigate the in vitro antitumor activity of L prior to initiating treatment on clinical trial. Results: With Anexin V staining for evaluation of apoptosis induction, in vitro testing of pts samples (n=10) showed only a modest increase in apoptosis at 200mg of L - levels clinically not achievable. Yet same pts treated with L on clinical study showed significant antitumor response, suggesting the mechanism to be possibly related to modulation of the tumor microenvironment. In evaluation of the tumor cytokine microenvironment (n= 10) we noted significant L induced increase in IL-10 (n=6), IL-8 (n=8), IP-10 (n=10), IL-8 (n=8), TNF-alpha (n=4) and decrease in PDGF (n=5) and RANTES (n=5). While evaluation of the immune cell repertoire we observed an absolute increase in T-cell as well as NK-cell after treatment with L. Conclusion: Our in vitro evaluation does not suggest a direct apoptotic effect of L on the malignant CLL cells and thus support the hypothesis that the anti-leukemic effect noted in the clinical trial (reported separately) is most likely from in vivo modulation of the tumor microenvironment as is demonstrated from changes in the cytokine milieu and the cellular immune response. Collectively these changes may be responsible for the immune modulating properties of L and the resultant anti-CLL activity in pts.

In various skeletal muscles, the mean density of acetylcholine receptors in the muscle postsynaptic membrane is constant at about 8700 per μm 2 , even though the overall size of an endplate ranged from 400 μm 2 to 1300 μm 2 in these muscles. This measurement was by electron microscope autoradiography of α-[ 3 H]bungarotoxin binding sites; only one-half of these, however, appear to be true active centers of the acetylcholine receptor. The highest density of these receptors is on the juxtaneuronal regions of the postsynaptic membrane, and their density in the depths of the fold is less than one quarter of that at the tips. A maximum sensitivity to externally applied acetylcholine, about 3000-5000 mV/nC, is found in diverse types of endplates when truly focal recording is achieved. This acetylcholine sensitivity appears to be determined by the local density of receptors in the membrane, and not by their total number at the endplate. A quantal efficiency term is also disclosed. The maximal sensitivity per molecule obtainable by microiontophoresis of acetylcholine is 5-10% of that operative when a quantum reacts. When acetylcholine is released from a vesicle, in contrast to its application from outside, geometric factors are more favorable. Consideration of the local packing density, acetylcholine molecule numbers, and the current flow when one quantum of acetylcholine interacts at the membrane suggests that one, or possibly two, activated receptor active centers are linked to one open gate of the ionic conductance modulator.

Tunicamycin was found to specifically inhibit the incorporation of a number of sugars into L1210 leukemia cell glycoproteins. This inhibition of glyco-protein biosynthesis led to a cessation of cell growth which was reversible in a dose-dependent and time-dependent manner. After removal of the antibiotic from L1210 cell cultures resumption of sugar incorporation preceded that of thymidine incorporation and the recovery of cell growth. The treatment of cells with tunicamycin resulted in a significant increase in the intracellular pool of UDP-N-acetylglucosamine which occurred concurrently with alterations in cell ultrastructure including distentions of the endoplasmic reticulum and nuclear membranes. Similar ultrastructural changes and increases in the intracellular pools of UDP-sugars were observed in L1210 cells exposed to 5 mM D-glucosamine, which suggested that the antiproliferative effects of tunicamycin may be related to the accumulation in the endoplasmic reticulum of one or more nucleotide sugar precursors of asparagine-linked glycoprotein biosynthesis. However, the biological effects of tunicamycin could be distinguished from those caused by D-glucosamine. Exposure of L1210 cells to tunicamycin resulted in specific alterations in the biochemical composition of the plasma membrane and in the inhibition of cellular agglutination by wheat germ agglutinin which were not apparent following exposure to equitoxic concentrations of the aminosugar. These studies, together with those which demonstrated that recovery of the cellular capacity to synthesize glycoproteins was obligatory for the recovery of cellular proliferation in tunicamycin-treated cells, suggested that inhibition of the synthesis of glycoproteins was the major factor limiting L1210 leukemic cell proliferation.

A novel synthesis of the nucleoside analog, 5′-deoxy-5′-(cyclopropylmethylthio)adenosine (CPMTA, 1) has been developed. CPMTA is a closely related structural analog of 5′-deoxy-5′-(isobutylthio)-adenosine (SIBA, 2), which has been widely studied and shown to exert a multitude of biological effects. The in vitro and in vivo antitumor (L1210 leukemia) activity of CPMTA has been found to be comparable to that of SIBA, whereas its in vitro antiviral (HSV and VSV) activity is diminished. These agents are being developed as inhibitors of methylation and/or polyamine synthesis.

Four variants of the cultured human cell line VA2 have been isolated which are resistant to the antiproliferative and antimitochondrial effects of methylglyoxal-bis(guanylhydrazone) (MGBG). Each of the four variants is two- to fivefold more sensitive to the mitochondrial protein synthesis inhibitor chloramphenicol (CAP) than wild type when grown in the absence of MGBG, and five- to tenfold more sensitive to CAP when grown in the presence of MGBG. Uptake studies demonstrate that each MGBG-resistant variant cell line is freely permeable to CAP. The in vivo rates of mitochondrial protein synthesis are significantly reduced in each of the variants whether pregrown and labeled in the presence or absence of MGBG. When cytoplasts from a cytoplasmically inherited CAP-resistant mutant are fused to an MGBG-resistant recipient cell line, cybrid clones can be isolated which are functionally resistant to low levels of CAP. With continued growth, the levels of resistance to CAP do not, however, approach the levels of resistance of the CAP-resistant donor cell line. When CAP resistance is subsequently transferred from a CAP/MGBG-resistant cybrid by enucleation and fusion to other human cell lines, then CAP-resistant cybrids can be readily selected in high levels of CAP. It is possible that the substantial decrease in mitochondrial protein synthesis observed in the variants fully accounts for their increased sensitivity to CAP, although the basis for this decreased rate of mitochondrial protein synthesis is not understood.

The analysis of naturally occurring variations in hormone-regulated gene expression generates insights into the mechanisms governing evolutionary changes in hormone response. In the mouse (genus Mus), kidney ornithine decarboxylase (ODC) expression, which is regulated by androgens, has been modified during evolution. This has resulted in intra- and interspecies variations that have accumulated over a 10-15 million year time period. We have examined ODC expression and its response to androgens in eight Mus species. Induced ODC levels were found to be similar in six of these species. Two species (M. cookii, M. pahari) contain diminished enzyme levels that are the result of different mechanisms. In M. cookii, the low ODC levels reflect reduced ODC mRNA induction in response to hormone. In M. pahari, on the other hand, the low ODC levels are not derived from altered mRNA concentrations, but appear to be due to translational and/or posttranslational effects. Nuclear run-on assays indicate that ODC mRNA induction is associated with increases in rates of ODC gene transcription. The low ODC mRNA level in induced M. cookii reflects a low rate of ODC gene transcription. Thus, both transcriptional and posttranscriptional mechanisms have contributed to modification of the ODC expression phenotype during evolution. The significance of these findings to the evolution of androgen-regulated ODC expression is discussed.

S-(5'-Deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA) is an irreversible inhibitor of S-adenosylmethionine (AdoMet) decarboxylase, which is designed to bind covalently the pyruvate residue at the enzyme active site. In the present study the cellular effects of AMA were characterized for the first time in cultured L1210 leukaemia cells. At the approximate IC50 (concn. giving 50% inhibition; 100 microM), AMA decreased spermidine and spermine by more than 80% at 48 h while increasing putrescine more than 10-fold. As an indication of enzyme specificity, growth inhibition was fully prevented with exogenous spermidine. When compared with the irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO), at similar growth-inhibitory concentrations, AMA was less cytotoxic, as determined by colony-formation efficiency. In combination with AMA, DFMO eliminated the rise in putrescine and decreased growth in an additive manner. The near-total depletion of intracellular polyamine pools achieved with the drug combination provided an opportunity to examine the relative abilities of individual polyamines to support growth and viability. Of the three exogenously supplied polyamines, only spermidine fully sustained cell growth and viability at control values during incubations totalling 120 h. By contrast, spermine supported growth at 23% of control and viability at 8%. Putrescine was similarly ineffective, supporting growth at 13% of control and viability at 7%. The data indicate that, in L1210 cells, spermidine is apparently the preferred polyamine in growth-related functions and is capable of fully supporting cell growth by itself. However, because spermine and putrescine can also support growth to some extent, maximum interference with growth and viability is best achieved by strategies which deplete all three polyamine pools.

The progress reviewed here would seem to validate the regulatory approach to interference with polyamine biosynthesis as an antiproliferative strategy. To our knowledge, this is the first example, among anticancer drugs, of pharmacological intervention of a biochemical pathway based strictly on regulatory control. Several features of polyamine biology naturally favor this approach and may account for its relative success. These include (a) the nature of the regulatory mechanisms themselves, (b) the exquisite sensitivity of the pathway to regulatory control, (c) the rapid turnover of ODC and AdoMetDC, (d) the different structural specificity of ODC and AdoMetDC regulation versus growth-dependent functions, and (e) the direct dependence of growth on sustained polyamine biosynthesis. As such, the regulatory approach to interference with polyamine biosynthesis offers several advantages over the use of specific enzyme inhibitors (Table 10). Of these, perhaps, the more significant are the facts that more than one enzyme can be simultaneously and specifically suppressed and that compensatory mechanisms, which otherwise counter the effects of enzyme inhibitors (11), are not invoked. We are encouraged by the concurrence of in vitro mechanistic findings with the predictions of the hypothesis for the regulatory approach and by the in vitro and in vivo growth inhibitory effects of the analogs against murine leukemia. One disadvantage of the regulatory analogs, such as BESm, has been that, as with specific polyamine inhibitors such as DFMO, analog-induced polyamine depletion results in cytostatic growth inhibition. While this response may help to minimize host toxicities, it clearly compromises antitumor activity. An intriguing exception to this generality has recently been found among human lung carcinoma cell lines. Previously, Luk et al. (93, 94) and others (95) reported that, among a spectrum of human lung carcinoma lines, small cell carcinoma was exquisitely sensitive to the ODC inhibitor, DFMO. Not only did these cells display a cessation of growth but also an inability to survive during DFMO-induced polyamine depletion. Studies extending these findings to long term maintenance therapy in human small cell lung carcinoma implants in athymic mice revealed sustained growth inhibition of the tumor for longer than one year (96). Casero et al. (97) now find that human large cell carcinoma, which is otherwise refractory to chemotherapeutic intervention, displays a cytotoxic response in vitro to polyamine depletion induced by BES or BESm but not by DFMO. These findings suggest cytotoxic susceptibility to polyamine depletion appears to be agent and cell type specific. Based on these leads, current and future efforts will attempt to identify and exploit these specificities.

Treatment of cultured L1210 cells with 1 mM-L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), a methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), produced a rapid and near-total depletion of AdoMet by 4 h. After this, the pools recovered to 60% of control by 48 h, apparently because of an increase in AdoMet synthetase activity. Both AdoMet depletion and the accompanying increase in synthetase activity were substantially enhanced by lowering methionine concentrations in the media from 100 microM to 30 microM, the minimal concentration that supports cell growth at control values. During a 4 h incubation in media containing 30 microM-methionine, 1-5 mM-L-cisAMB depleted cellular AdoMet to undetectable values, and inhibited nucleic acid methylation by 44-72% and RNA methylation by 60-87%. Under these same treatment conditions, putrescine pools increased by about 3-fold, whereas spermidine pools decreased by only 20% and spermine pools remained the same. Pool changes were accompanied by a 2-4-fold increase in ornithine decarboxylase activities and AdoMet activities. Thus the rapid depletion of AdoMet pools by L-cisAMB results immediately in a decrease in methyl-transfer reactions involving nucleic acids, whereas, by contrast, biosynthesis of higher polyamines appears to be minimally affected, owing to compensatory increases in key enzyme activities.

We have tested the notion that polyamines, particularly putrescine, mediate the response of mouse kidney to androgens. Hormonal effects were measured in female mice maintained on the ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO), which results in a 85-90% reduction of ODC enzyme levels and a depletion of putrescine concentrations in kidney. These animals exhibited normal kidney cell hypertrophy in response to testosterone. In addition, androgen-inducibility of the RP2 gene was indistinguishable from that in normal mice. These results indicate that an increase in putrescine levels is not a prerequisite for androgen effects in mouse kidney and that putrescine does not mediate the hormonal response.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.

1. 1. The interactions of naturally occurring polyamines: putrescine, spermidine and spermine, with anticancer bis-guanylhydrazones: methylglyoxal-bis(guanylhydrazone) (MGBG) and 4,4′-diacetyldiphenylurea-bis(guanylhydrazone) (DDUG) were investigated at the level of mitochondrial membrane. 2. 2. The effects of bis-guanylhydrazones on intact rat liver mitochondria were readily prevented or reversed by polyamines and these interactions were also affected by the mictochondrial transmembrane potential. 3. 3. Magnesium cations enhanced the protective action of polyamines. The data indicate that competition exists between the essential anticancer bis(guanylhydrazones) and polyamines for low affinity negatively charged binding sites at the outer surface of inner mitochondrial membrane. 4. 4. The study of drug interactions was extended to the level of isolated tumor mitochondria from rat HTC hepatoma and muraine L1210 leukemia cells. 5. 5. A complicated pattern of interactions between the anticancer bis-guanylhydrazones and phenethylbiguanide was obtained.

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 greater than polyps greater than 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.

Intact murine L1210 leukemic cells incorporated significant quantities of [3H]-N-acetylneuraminic acid directly from CMP-N-acetylneuraminic acid. When pretreated with Vibrio cholerae neuraminidase, incorporation increased sixfold to tenfold. Biochemical studies comparing incorporation of N-acetyl-neuraminic acid from the nucleotide sugar with that from free sugar demonstrated that the relatively high levels of incorporation from CMP-N-acetyl-neuraminic acid could not be due to the incorporation of free sugar generated by extracellular degradation of the nucleotide sugar. Very little N-acetylneuraminic acid was taken up or incorporated by L 1210 cells from free sugar and this incorporation was not increased by neuraminidase pretreatment. Moreover, extracellular breakdown of CMP-N-acetylneuraminic acid during incubations with L 1210 cells was rather insignificant. Electron microscope autoradiography of cells incubated with CMP-N-acetylneuraminic acid demonstrated that greater than 84% of the incorporated radioactivity was associated with the plasma membrane and less than 1% with the Golgi apparatus. These findings are consistent with the conclusion that incroporation of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid is the consequence of a cell surface sialytransferase system. Pretreatment of cells with the nonpenetrating reagent, diazonium salt of sulfonilic acid, significantly inhibited this ectoenzyme system while only marginally affecting galactose uptake and incorporation at the Golgi apparatus. Interestingly, incorporation from CMP-N-acetylneuraminic acid declined as the viability of the cell population declined. When taken together, the above evidence develops a rigorous argument for the presence of a sialyltransferase enzyme system at the cell surface of L 1210 cells. Studies directed towards the detection of a similar ectogalactosyltransferase system were also undertaken. Cells incubated in the presence of UDP-[3H]-galactose incorporated radioactivity into a macromolecular fraction. The presence of excess unlabeled galactose in the incubation medium significantly reduced this incorporation. Electron microscope autoradiographs of cells incubated with UDP-[3H]-galactose, demonstrated that incorporation occurred primarily at the Golgi apparatus. The grain distribution in these autoradiographs was similar to that for free galactose. Thus, the incorporation observed for L-1210 cells incubated in UDP-[3H]-galactose was due primarily to the intracellular utilization of free galactose generated by extracellular degradation of the nucleotide sugar. Inability t o demonstrate an ectogalacto-syltransferase system on L1210 cells does not rule out the possibility that the enzyme is present but undetectable due t o the absence of appropriate cell surface acceptor molecules.

Twelve different analogs of spermidine (SPD) and SPD itself were compared for their ability to modulate two conformational transitions of DNA; the B-to-Z conformational transition of poly(dG-me5dC) and the thermal melting transition of calf thymus DNA. The analogs consisted of five N-ethyl-SPD derivatives [N1-ethyl-SPD, N4-ethyl-SPD, N8-ethyl-SPD, N1,N8-bis(ethyl)SPD and N1,N4,N8-tri(ethyl)SPD], which differed in the number and/or position of the ethyl substitution (the alkyl series); three N-acetyl-SPD derivatives (N1-acetyl-SPD, N4-acetyl-SPD, and N8-acetyl-SPD), which were comparable to the N-ethyl-SPD derivatives but not protonated at the substituted amine (the acyl series); three aliphatic analogs [nor-SPD, homo-SPD, and N1,N9-bis(ethyl)homo-SPD], which differed in the interamine carbon chain length (homolog series), and 1,8-diaminooctane, which was comparable in overall chain length to SPD but lacked a central nitrogen. By comparing the relative abilities of the various analogs and SPD to modulate DNA structural transitions, it is possible to gain insight into the relative significance of the number and location of protonated amines (acyl series), the number and location of steric groups (alkyl series), aliphatic chain length (homolog series), and the central amine (1,8-diaminooctane) as determinants of SPD–DNA interactions. The B-to-Z conformational transition was facilitated to a midpoint by 2.4 μM SPD under conditions of low (i.e., 11 mM Na+) ionic strength. The phenomenon was affected most significantly by the number of protonated amines followed in rank order by location of the protonated amines, number of steric groups (bulk), steric group location, and aliphatic chain length. Stabilization of DNA to thermal melting was also most affected by the number of protonated amines followed by aliphatic chain length, number of steric groups, and location of protonated amines. In general, substitutions at the central (N4) amine of SPD exerted a significant influence on the B-to-Z transition but not on thermal melting.

Polyamine oxidase from L1210 cells has been separated into two isozymes, A and B on the basis of chromatography on hydroxyapatite. The two isozymes also show different elution characteristics from MGBG Sepharose and Sephacryl-300. The molecular weights of A and B isozymes are estimated to be 260,000 and 200,000 daltons, respectively. The two isozymes show different kinetic characteristics with spermidine, spermine, N1-acetylspermine, and N1,N12-diacetylspermine.

Histochemical techniques were used successfully to investigate the oxidative and hydrolytic enzymes of a Cotylocercous cercaria tentatively identified as the larva of Plagioporus lepomis Dobrovolny. In tests for 11 hydrolytic enzymes, nonspecific esterase was detected in the nervous system, leucine aminopeptidase and acid phosphatase activities were found in the excretory system, and three phosphatases and phorylase were located in the subcuticular area, in the caudal region, or in the suckers. The possible functions of these enzymes are discussed with respect to their location. Results of tests for 13 oxidative enzymes suggest the presence of a glycolytic pathway, pentose phosphate pathway, Kreb's cycle, respiratory chain, and lipid synthesis. Sites of intensive oxidative enzyme activity include the subcuticular area, oral and ventral suckers, nervous system, and parenchyma.

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 μM 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.

Purpose To understand the mechanisms behind platinum drug/DENSPM-induced inhibition of cancer cell growth, we compared the effects of oxaliplatin and cisplatin when combined with DENSPM on the induction of SSAT mRNA, activity, polyamines and cell growth in A2780 human ovarian carcinoma cells and their oxaliplatin- and cisplatin-resistant variants A2780/C10B and A2780/CP, respectively. Methods Parental and Pt-resistant cells were treated with platinum agent alone, DENSPM alone or combination (10 μM each, 20 h). QRT–PCR, radioactive product measurement and HPLC were used for mRNA, activity and polyamine pools, respectively; drug interaction on cell growth was by SRB and isobologram analysis. Results Both platinum agents induced SSAT mRNA in parental A2780 cells, but not in resistant cells. Platinum drug/DENSPM combinations produced high levels of SSAT activity in parental cells with significant depletion of spermine and spermidine, but not in resistant cells. Co-treatment with platinum agents increased the levels of DENSPM in all cell lines. Oxaliplatin/DENSPM combination was superior to cisplatin/DENSPM in the inhibition of cell growth in parental cells. No synergy was observed in the resistant cells. Conclusions Increased DENSPM levels following co-treatment with Pt agents enhances the translation and stability of SSAT protein leading to polyamine pool depletion, facilitating more Pt–DNA adduct formation in parental cells. Oxaliplatin/DENSPM combination is superior to cisplatin/DENSPM in cell growth inhibition as DACH-Pt DNA adducts are cytotoxic even at relatively fewer numbers. Reduced platinum uptake in Pt-resistant cells contributes to reduced SSAT mRNA induction and absence of synergy when combined with DENSPM.

Six structural homologs of spermidine and five of its precursor, putrescine, were studied for their ability to prevent cytostasis of cultured L1210 leukemia cells induced by alpha-difluoromethylornithine (DFMO), a specific inhibitor of putrescine biosynthesis. High-performance liquid chromatography and competition studies with spermidine indicated that the homologs, which vary in the length of the carbon chain separating the amines, penetrated the cells. The structural specificity of the spermidine carrier was defined. Three of the six spermidine homologs supported cell growth during a 48-hour incubation in the presence of DFMO, indicating that a two-carbon extension of spermidine structure was tolerated for biological function. Two of the five putrescine homologs supported growth after being converted by the cells to their respective spermidine homologs. The central nitrogen of spermidine appears to be essential for function since diamines of chain length comparable to that of spermidine did not prevent DFMO cytostasis. No more than 15 percent of the spermidine normally present in L1210 cells was required for cell proliferation in the presence of DFMO.

In attempting a molecular analysis of the events involved in the chemical transmission of an impulse, we have selected the vertebrate motor endplate as a model synapse. Ultrastructural localization of certain functional molecules involved there reveals that the acetylcholine (ACh) receptor is concentrated at the crests of the postjunctional folds at a constant density of 20,000–25,000/μm2 of membrane while, by contrast, cholinesterase sites are distributed evenly along the folds. This evidence, together with that from other laboratories, has led to the construction of an endplate model showing the spatial arrangement of transmitter release in relation to ACh receptor and acetylcholinesterase concentrations. By several approaches, the end-plates of different species and muscle types are found to have a fixed number of ACh receptors ranging from 107 to 108 per endplate. Of these, over 90% are now considered to be true ACh receptors as based on their affinity for d-tubocurarine. Interestingly, the maximum sensitivity values for ACh, applied microiontophoretically to these same muscle types, all tend to be in the same range—5000 to 8000 mv/nc. It therefore appears that the endplate response to ACh is determined by the density of receptors along the post-junctional membrane and not by the total number at the endplate. From several considerations, we deduce that this density of receptor sites cannot be saturated by ACh during normal transmission. The correlation of electrophysiological data with quantitative molecular parameters, from these and other laboratories, indicates that the maximum endplate sensitivity to externally applied ACh (∼ 7000 mv/nc) is equivalent to an efficiency of about 12% of that of neurally released transmitter. By similar analysis, a one-to-one or two-to-one relationship for the ACh receptor and the ionic conductance modulation system has been derived, implying that the two may be linked as such in the membrane. ACh receptor, having an apparent molecular weight of about 370,000 (in 0.2% Triton X-100), has been solubilized and purified from denervated muscle.

Chinese hamster ovary (CHO) cells in culture were limited for polyamines through the use of alpha-methylornithine (alpha MO), a competitive inhibitor of ornithine decarboxylase. Initial exposure of the cells to the inhibitor caused growth rate and intracellular polyamine content to decline continuously. Reseeding the alpha MO-treated cells into medium containing the inhibitor resulted in steady-state (exponential) growth at cell densities below 5 x 10(3) cells/cm2, at a rate approximately twofold slower than untreated cells. Under these conditions, putrescine and spermidine were undetectable and spermine remained relatively constant at a level approximately half that found in untreated cells. Addition of exogenous putrescine elevated the polyamine content and stimulated the growth of alpha MO-treated cultures. Thus, growth rate correlated with polyamine content in the alpha MO-treated cells. The growth of reseeded, alpha MO-treated cells became nonexponential at a density (5 x 10(3) cells/cm2) far below that at which untreated cells departed from exponential growth (1 x 10(5) cells/cm2). Medium obtained from high density, alpha MO-treated cultures inhibited the growth of cells at low density in the presence of alpha MO. Doubling the concentration of the defined components of conditioned medium did not markedly affect its capacity to inhibit growth. However, dialysis completely not markedly affect its capacity to inhibit growth. However, dialysis completely removed the inhibitory activity from conditioned medium. The results imply that a low molecular weight inhibitor of growth is produced by polyamine-limited cells. This is a variable that must be controlled in studies with polyamine-limited animal cells. Morphological studies indicated that subcellular organelles, including mitochondria, were largely unaffected by treatment with alpha MO. The maintenance of mitochondrial integrity in the presence of alpha MO demonstrates that the swelling of mitochondria observed previously in cells treated with methylglyoxal bis(guanylhydrazone) was not due to polyamine limitation. alpha MO-treated cells did, however, accumulate numerous cytoplasmic vacuoles. The identity of these vacuoles and their relationship to cellular physiology is not yet understood.

The abilities of the naturally occuring polyamines, putrescine, spermidine and spermine, to affect variables related to the bioenergetic functions of isolated rat liver mitochondria were studied. At concentrations comparable to those present intracellularly, the polyamines inhibited state 4 respiration, but they had much less effect on state 3 or uncoupled respiration. The concentrations required to produce 25% inhibition (I25) of state 4 respiration varied according to the polyamine, with putrescine being least effective (I25, 20mM) and spermidine and spermine being more effective and comparable (I25, 7.5 and 7.0mM respectively). This inhibition was antagonized by 15 mM potassium and enhanced by valinomycin and 4 mM magnesium. Inhibition of monoamine oxidase, an enzyme of outer mitochondrial membrane, was also observed to occur. Addition of polyamines to mitochondrial suspensions caused an increase in the optical density and protected against the swelling effects of sublytic concentrations of Triton X-100. By electron microscopy, polyamines were found to cause the outer mitochondrial compartment to collapse bringing the inner and outer membranes into apparent contact with one another. The electrophoretic mobility of mitochondria toward the anode was markedly slowed by polyamines (i.e. 50% by 1.25 mM spermine), indicating surface binding and neutralization of the negative surface charge. In almost all of the above mitochondrial effects, spermine and spermidine were similar in effectiveness and putrescine was less effective. It is suggested that polyamines may be capable of modulating respiration of isolated mitochondria by binding to non-specific anionic sites at the surface of the inner mitochondrial membrane. Neutralization of the net negative surface potential may interfere with cation fluxes across the membrane, particularly those of potassium.

Publisher Summary The changes in the biochemical properties of plasma membrane components are known to occur following the oncogenic transformation of cells. Many of these changes specifically involve the carbohydrate portion of cell surface glycoconjugate molecules. Thus, a selective chemotherapeutic inhibition of tumor cell growth might be achieved with the analogs of membrane sugars. These sugar analogs may act on tumor cells by interfering with the synthesis, structure, function, and/or immunogenicity of membrane glycoconjugates. This chapter discusses the synthesis of the halogenated analogs of L-fucose (I and II) and of D-galactose (III). It presents a table to show the cytotoxicity of L-Fucose analogs at 1 mM.

Four methionine analog inhibitors of methionine adenosyltransferase, the enzyme which catalyzes S-adenosylmethionine biosynthesis, were tested in cultured L1210 cells for their effects on cell growth, leucine incorporation, S-adenosylmethionine (AdoMet) formation and polyamine biosynthesis. The IC50 values were as follows: selenomethionine, 0.13 mM; L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cis-AMB), 0.4 mM; cycloleucine, 5 mM and 2-aminobicyclo[2.1.1]hexane-2-carboxylic acid, 5 mM. At IC50 levels, the analogs significantly reduced AdoMet pools by approximately 50% while not similarly affecting leucine incorporation or polyamine biosynthesis. In combination with inhibitors of polyamine biosynthesis, growth inhibition was greatly increased with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase, but only slightly increased with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Overall, the data indicate that the methionine analogs, and particularly L-cis-AMB, seem to inhibit cell growth by interference with AdoMet biosynthesis. Since polyamine biosynthesis is not affected, the antiproliferative effect may be mediated through perturbations of certain transmethylation reactions.

Our current rationale for targeting polyamines as a mechanism-based drug discovery initiative is based on several diverse yet interrelated considerations (Table 1) some of which derive from findings to be reviewed here. Of these, the most critical are (a) the strict dependence of proliferating cells on polyamine availability (b) the increased rate of polyamine biosynthesis and transport associated with neoplastic tissues relative to their normal counterparts and (c) the novelty of polyamine biosynthesis and/or function as a departure from more cyto-toxic DNA-directed approaches. While admittedly, an absolute basis for selectivity has not yet been defined, recognition must be given to the fact that no chemotherapeutic strategy described to date is wholly selective for tumor tissue and yet total curability of a number of malignant diseases can be achieved. We believe this justifies the evaluation of new and novel approaches even in the absence of absolute metabolic rationale for selective tumor cell kill. Although still confined to animal model systems, encouraging antitumor data with polyamine analogs lend credence to this belief (Bergeron et al., 1988).

The localization and quantitation of the acetylcholine receptor at the normal and 5-day denervated mouse diaphragm endplate has been evaluated by means of labeling with tritiated α-bungarotoxin followed by electron microscope autoradiography. The toxin binds specifically and irreversibly to the acetylcholine receptor molecules of skeletal muscle. The distribution of silver grains over denervated endplates labeled to saturation with this toxin is consistent with a receptor location on the postjunctional membrane at a mean density similar to that of the innervated endplate, 8,500/μm 2 . As in the innervated endplate, the receptors are not located uniformly along the membrane, but are concentrated at the fold crests, i.e., that area nearest to the axonal membrane when it is present, at a density near to 22,000/μm 2 . Since morphometric measurements show that the total postjunctional membrane at the denervated endplate is unchanged at 5 days, we deduce from the mean receptor site density that the total receptor population at the endplate remains the same. On the basis of these findings, we conclude that only a very small population of nicotinic receptors could be associated with the terminal axon, and perhaps that none at all are present there. Further, the receptors on the postjunctional membrane are unaffected in either density or distribution following the removal of neural influences by denervation even though these same influences have a profound effect on extrajunctional receptors at later periods of denervation were not possible due to degenerative tissue changes associated with denervation.