Polyamine biosynthesis impacts cellular folate requirements necessary to maintain S-adenosylmethionine and nucleotide pools.

Folate (vitamin B9) is utilized for synthesis of both S-adenosylmethionine (AdoMet) and deoxythymidine monophosphate (dTMP), which are required for methylation reactions and DNA synthesis, respectively. Folate depletion leads to an imbalance in both AdoMet and nucleotide pools, causing epigenetic and genetic damage capable of initiating tumorigenesis. Polyamine biosynthesis also utilizes AdoMet, but polyamine pools are not reduced under a regimen of folate depletion. We hypothesized that high polyamine biosynthesis, due to the high demand on AdoMet pools, might be a factor in determining sensitivity to folate depletion. We found a significant correlation (P<0.001) between polyamine biosynthesis and the amount of folate required to sustain cell line proliferation. We manipulated polyamine biosynthesis by genetic and pharmacological intervention and mechanistically demonstrated that we could thereby alter AdoMet pools and increase or decrease demand on folate availability needed to sustain cellular proliferation. Furthermore, growing a panel of cell lines with 100 nM folate led to imbalanced nucleotide and AdoMet pools only in cells with endogenously high polyamine biosynthesis. These data demonstrate that polyamine biosynthesis is a critical factor in determining sensitivity to folate depletion and may be particularly important in the prostate, where biosynthesis of polyamines is characteristically high due to its secretory function.

Polyamine metabolism and tumorigenesis in the Apc(Min/+) mouse.

While polyamine homoeostasis is clearly important in maintenance of normal cell function, the roles of these cations, as well as the enzymes that regulate their metabolism, in the neoplastic process are not clear. In particular, the polyamine catabolic enzyme SSAT (spermidine/spermine N(1)-acetyltransferase) seems to have different roles in tumorigenesis, depending upon the particular system being analysed. In attempts to clarify the function of SSAT in tumour development, we have utilized the Apc(Min/+) mouse, which carries a mutant allele of the Apc (adenomatous polyposis coli) gene, rendering it susceptible to the formation of multiple adenomas in the small intestine and colon. Using genetically engineered animals (i.e. transgenic and knockout mice), we have shown that SSAT acts as a tumour promoter in the Apc(Min/+) model. Modulation of tumorigenesis is not associated with changes in tissue levels of either spermidine or spermine. These findings, along with those made in other animal models of cancer, have prompted us to propose that metabolic flux through the polyamine biosynthetic and catabolic pathways, and the consequent changes in levels of various metabolites within the cell (i.e. the metabolome), is critical to tumour development. The metabolic flux model represents a novel way of thinking about the role of polyamines in cell physiology and the neoplastic process.

Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAF1/CIP1/SDI1, changes in the expression of p21-regulated genes, and a senescence-like phenotype.

The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment of MALME-3M cells with either the ornithine decarboxylase inhibitor alpha-difluoromethylornithine or the S-adenosylmethionine decarboxylase inhibitor MDL-73811 lowered specific polyamine pools and slowed cell growth but did not induce cell cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G1 arrest. The latter was associated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma protein. All effects were fully prevented by exogenous polyamines. p21 induction preceded p53 stabilization in MALME-3M cells and also occurred in a polyamine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 is induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to inhibit the expression of multiple proliferation-associated genes and to induce the expression of genes associated with various aspects of cell senescence and organism aging. Polyamine depletion in MALME-3M cells was associated with inhibition of seven of seven tested p21-inhibited genes and with induction of 13 of 14 tested p21-induced genes. p21 expression is also known to induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in size, appeared more granular, and expressed senescence-associated beta-galactosidase. Cells released from the polyamine inhibition lost the ability to form colonies, failed to replicate their DNA, and approximately 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induction in fibrosarcoma cells. The results of this study indicate that polyamine pool depletion achieved by specific biosynthetic enzyme inhibitors causes p21-mediated G1 cell cycle arrest followed by p21-mediated changes in gene expression, development of a senescence-like phenotype, and loss of cellular proliferative capacity.

Apoptotic signaling in polyamine analogue-treated SK-MEL-28 human melanoma cells.

N(1),N(11)-Diethylnorspermine (DENSPM) is a polyamine analogue with clinicalrelevance as an experimental anticancer agent and the ability to elicit a profound apoptotic response in certain cell types. Here, we characterize the polyamine effects and apoptotic signaling events initiated by treatment of SK-MEL-28 human melanoma with 10 microM DENSPM. Maximal induction of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine pool depletion were seen by 16 h, whereas early apoptosis was first apparent at 36 h. Intermediate events related to apoptotic signaling were sought between 16 and 36 h. A loss of mitochondrial transmembrane potential (Deltapsi(m)) beginning at 24 h was followed by the release of cytochrome c into the cytosol at 30 h. Loss of mitochondrial integrity was accompanied by caspase-3 activation and poly(ADP-ribose) polymerase digestion from 30 to 36 h. The caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone rendered cells resistant to analogue-induced caspase-3 activation and reduced the apoptotic response in a dose-dependent manner. Because polyamine reduction achieved by inhibitors of polyamine biosynthesis inhibited growth but did not cause apoptosis, we looked for alternative polyamine-related events, focusing on induction of SSAT. Three DENSPM analogues that differentially induced SSAT activity but similarly depleted polyamine pools revealed a close correlation between enzyme induction and cytochrome c release, caspase activation, and apoptosis. Dose-dependent inhibition of polyamine oxidase, an enzyme that oxidizes acetylated polyamines generated by SSAT and releases toxic by-products such as H(2)O(2) and aldehydes, prevented cytochrome c release, caspase activation, and apoptosis. Taken together, the findings indicate that DENSPM-induced apoptosis is at least partially initiated via massive induction of SSAT and related oxidative events and subsequently mediated by the mitochondrial apoptotic signaling pathway as indicated by cytochrome c release and caspase activation.

Post-transplant lymphoproliferative disease in children.

Epstein-Barr virus (EBV)-driven post-transplant lymphoproliferative disease (PTLD) is an important cause of morbidity and mortality following transplantation, and it occurs more frequently in children than in adults. Of 22 (5%) children at our institution who developed tissue-proven PTLD 1-60 months (mean 16.5 months) following organ transplant, 11 died: nine of these 22 patients developed PTLD between 1989 and 1993, and seven (78%) died; the remaining 13 developed PTLD between 1994 and 1998, and four (31%) died (p = 0.08). All nine patients who developed PTLD < 6 months after transplant died, but 11 of 13 patients who manifested disease > or = 6 months after transplant survived (p = 0.0002). Ten of 11 (91%) survivors, but only two of eight (25%) children who died, had serologic evidence of EBV infection at the time of PTLD diagnosis (p = 0.04). EBV seroconversion identified patients at risk for developing PTLD, but also characterized patients with sufficient immune function to survive EBV-related lymphoid proliferation. In situ hybridization for EBER1 mRNA was diagnostically helpful because it detected EBV in tissue sections of all 20 patients with B-cell PTLD, including those with negative serology.

Birth weight variation in the domestic pig: effects on offspring survival, weight gain and suckling behaviour.

In domestic pigs, litter-mates often vary considerably in birth weight. To examine whether this size variation influences piglet survival, weight gain and suckling behaviour, we experimentally manipulated the number and size distribution of litter-mates in 51 litters. Litters were small (eight or nine piglets) or large (11 or 12 piglets) compared to the herd mean of 10 piglets, and were made more or less variable in weight by using the largest and smallest quartiles of two combined litters (variable) or the middle two quartiles (uniform). Weights were measured on days 0, 3 and 21. Behavioural measures (percent of nursings missed, mean teat consistency score, per capita number of teat disputes before milk ejection, and percent time spent in teat disputes in the 20min after milk ejection) were recorded on days 1, 4, 10 and 17. Piglet weight variation (percent of coefficient of variation, CV) almost doubled over the 21 days in uniform litters and actually decreased in variable litters, but still remained higher in the variable litters. Overall, survival, percent of nursings missed, consistency in piglets' use of teats, number of teat disputes, percent time a piglet spent in teat disputes after milk ejection, and weight gain were unaffected by birth weight variation although there was a tendency (P=0.09) for more piglet deaths in variable litters. Behavioural measures of sibling competition were higher in large litters. The data provide little support for the hypotheses that high birth weight variation results in decreased survival, or that it permits rapid establishment of dominance, thereby reducing wasteful competitive behaviour in surviving piglets.

Torpor and digestion in food-storing hibernators.

Many species of hibernating mammals rely on hoarded food rather than body fat to support winter energy requirements. Here, we evaluate whether the associated ingestive and digestive requirements reduce the benefits that food-storing hibernators can accrue from torpor. Using a simple model, we predict (1) that digestive efficiency could either increase or decrease with increased use of torpor, depending on the Q(10) of digestion relative to the Q(10) of whole-animal metabolism and (2) that increased torpor will result in a linear decrease in energy consumption but an exponential increase in euthermic intake requirements. In 16 captive eastern chipmunks (Tamias striatus), the proportion of time that different individuals spent in torpor was highly variable (29.8%+/-5.9%; 0.0%-86.3%), positively correlated with dry matter digestibility (r2=0.53, P=0.02) and negatively correlated with energy consumption (r2=0.72, P=0.002). Thus, by both increasing conversion efficiency and reducing energy requirements, torpor appears to provide a double benefit for energy conservation by food-storing hibernators. Despite this, a comparative analysis shows that the euthermic intervals of food-storing rodents are four times as long and torpor intervals are half as long as that of fat-storing rodents. Given that required euthermic intake rates are expected to increase exponentially at high levels of torpor, the reduced torpor expression of food-storing species may result from constraints on their ability to load enough food into the gut when euthermic to cover the energy requirements of the subsequent torpor cycle.

Serine/threonine phosphorylation of IRS-1 triggers its degradation: possible regulation by tyrosine phosphorylation.

Insulin receptor substrate (IRS)-1 protein expression is markedly reduced in many insulin-resistant states, although the mechanism for this downregulation is unclear. In this study, we have investigated the early events in the insulin pathway that trigger the degradation of IRS-1. Incubation of the adipocytes with insulin induced a fast electrophoretic mobility shift of IRS-1 and a subsequent degradation of the protein. Wortmannin and rapamycin blocked this mobility shift of IRS-1, maintained the insulin-induced tyrosine phosphorylation of IRS-1, and blocked its degradation. In contrast, a glycogen synthase kinase 3 inhibitor, a mitogen-activated protein kinase/extracellular-regulated kinase inhibitor, and various protein kinase C inhibitors had no effect. Incubation with okadaic acid increased the serine/threonine phosphorylation of IRS-1 and its degradation, mimicking insulin, and its effect was prevented by the proteasome inhibitor lactacystin, as well as by rapamycin. Treatment of the cells with the tyrosine phosphatase inhibitor orthovanadate in the presence of insulin or okadaic acid partially inhibited the degradation of IRS-1. We propose that a rapamycin-dependent pathway participates as a negative regulator of IRS-1, increasing its serine/threonine phosphorylation, which triggers degradation. Thus, regulation of serine/threonine versus tyrosine phosphorylation may modulate IRS-1 degradation, affecting insulin sensitivity.

[Phosphinic analog of methionine inhibits growth of leucosis cell L1210 and transforms to phosphinic analog of S-adenosylmethionine]. / Fosfinovyi analog metionina tormozit rost leikoznykh kletok L1210 i prevrashchaetsia v fosfinovyi analog S-adenozilmetionina.

A phosphinic analogue of methionine bearing a phosphinic H(OH)(O)P fragment in place of the carboxyl group inhibited the growth of the L1210 cells and was intracellularly transformed to the phosphinic analogue of S-adenosylmethionine.

Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs.

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.

Treatment of cells with the polyamine analog N, N11-diethylnorspermine retards S phase progression within one cell cycle.

When Chinese hamster ovary cells were seeded in the presence of the spermine analog N1,N11-diethylnorspermine (DENSPM), cell proliferation ceased; this was clearly apparent by cell counting 2 days after seeding the cells. However, 1 day after seeding there was a slight difference in cell number between control and DENSPM-treated cultures. To investigate the reason for this easily surpassed slight difference, we used a sensitive bromodeoxyuridine/flow cytometry method. Cell cycle kinetics were studied during the first cell cycle after seeding cells in the absence or presence of DENSPM. Our results show that DENSPM treatment did not affect the progression of the cells through G1 or the first G1/S transition that took place after seeding the cells. The first cell cycle effect was a delay in S phase as shown by an increase in the DNA synthesis time. The following G2/M transition was not affected by DENSPM treatment. DENSPM treatment inhibited the transient increases in putrescine, spermidine, and spermine pools that took place within 24 h after seeding. Thus, in conclusion, the first cell cycle phase affected by the inhibition of polyamine biosynthesis caused by DENSPM was the S phase. Prolongation of the other cell cycle phases occurred at later time points, and the G1 phase was affected before the G2/M phase.

Placement of pedicle screws in the human cadaveric cervical spine: comparative accuracy of three techniques.

STUDY DESIGN: This investigation was conducted in two parts. In the first part, a morphometric analysis of critical cervical pedicle dimensions were measured to create guidelines for cervical pedicle screw fixation based on posterior cervical topography. In the second part of the study, a human cadaver model was used to assess the accuracy and safety of transpedicular screw placement in the subaxial spine using three different surgical techniques: 1) using surface landmarks established in the first part of the study, 2) using supplemental visual and tactile cues provided by performing laminoforaminotomies, and 3) using a computer-assisted surgical guidance system. OBJECTIVE: To assess the accuracy of transpedicular screw placement in the cervical spine using three surgical techniques. SUMMARY OF BACKGROUND DATA: A three-column fixation device implanted to secure an unstable cervical spine can be a valuable tool with a biomechanical advantage in the spine surgeon's armamentarium. Despite this advantage, concerns over surgical neurovascular complications have surfaced. Cadaver-based morphometric measurements used to guide the surgeon in the placement of a pedicle screw show significant variability, raising legitimate concerns as to whether transpedicular fixation can be applied safely. METHODS: Precise measurements of 14 human cadaveric cervical spines were made by two independent examiners of pedicle dimensions, angulation, and offset relative to the lateral mass boundaries. On the basis of this analysis, guidelines for pedicle screw placement relative to posterior cervical topography were derived. In the second part of the study, 12 human cadaveric cervical spines were instrumented with 3.5-mm screws placed in the pedicles C3-C7 according to one of three techniques. Cortical integrity and neurovascular injury were then assessed by obtaining postoperative computed tomography scans (1-mm cuts) of each specimen. Cortical breaches were classified into critical or noncritical breaches. RESULTS: Linear measurements of pedicle dimensions had a wide range of values with only fair interobservercorrelation. Angular measurements showed similarangulation in the transverse plane (40 degrees ) at each level. With respect to the sagittal plane, both C3 and C4 pedicles were oriented superiorly relative to the axis of the lateral mass, whereas the C6 and C7 pedicles were oriented inferiorly. The dorsal entry point of the pedicle on the lateral mass defined by transverse and sagittal offset had similar mean values with wide ranges, although there often was excellent correlation between observers. There were no significant interlevel, right/left, or male/female differences noted with respect to offset. Using one of three techniques, 120 pedicles were instrumented. In group 1 (morphometric data): 12.5% of the screws were placed entirely within the pedicle; 21.9% had a noncritical breach; and 65. 5% had a critical breach. In group 2 (laminoforaminotomy), 45% of the screws were within the pedicle; 15.4% had a noncritical breach; and 39.6% had a critical breach. In group 3 (computer-assisted surgical guidance system), 76% of the screws were entirely within the pedicle; 13.4% had a noncritical breach; and 10.6% had a critical breach. Regardless of the technique used, the vertebral artery was the structure most likely to be injured. CONCLUSIONS: On the basis of the morphometric data, guidelines for cervical spine pedicle screw placement at each subaxial level were derived. Although a statistical analysis of cadaveric morphometric data obtained from the cervical spine could provide guidelines for transpedicular screw placement based on topographic landmarks, sufficient variation exists to preclude safe instrumentation without additional anatomic data. Insufficient correlation between different surgeons' assessments of surface landmarks attests to the inadequacy of screw insertion techniques in the cervical spine based on such specific topographic guide

The polyamine oxidase inhibitor MDL-72,527 selectively induces apoptosis of transformed hematopoietic cells through lysosomotropic effects.

Polyamine oxidase functions in the polyamine catabolic pathway, converting N1-acetyl-spermidine and -spermine into putrescine (Put) and spermidine (Spd), respectively, thereby facilitating homeostasis of intracellular polyamine pools. Inhibition of polyamine oxidase in hematopoietic cells by a specific inhibitor, N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL-72,527), reduces the levels of Put and Spd and induces the accumulation of N1-acetylated Spd. Although previously thought to be relatively nontoxic, we now report that this inhibitor overrides survival factors to induce cell death of several immortal and malignant murine and human hematopoietic cells, but not of primary myeloid progenitors. Cells treated with MDL-72,527 displayed biochemical changes typical of apoptosis, and cell death was associated with the down-regulation of the antiapoptotic protein Bcl-X(L). However, enforced overexpression of Bcl-X(L), or treatment with the universal caspase inhibitor zVAD-fmk, failed to block MDL-72,527-induced apoptosis in these hematopoietic cells. Despite decreases in Put and Spd pools, MDL-72,527-induced apoptosis was not blocked by cotreatment with exogenous Put or Spd, nor was it influenced by overexpression or inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase. Significantly, MDL-72,527-induced apoptosis was associated with the rapid formation of numerous lysosomally derived vacuoles. Malignant leukemia cells were variably sensitive to the lysosomotropic effects of MDL-72,527, yet pretreatment with the ornithine decarboxylase inhibitor L-alpha-difluoromethylornithine sensitized all of these leukemia cells to the deleterious effects of the inhibitor by stimulating its intracellular accumulation. The lysosomotropic nature of select polyamine analogues may, thus, provide a novel chemotherapeutic strategy to selectively induce apoptosis of malignant hematopoietic cells.

Transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N1-acetyltransferase show enhanced sensitivity to the polyamine analog, N1, N11-diethylnorspermine.

We have recently generated transgenic mice in which polyamine catabolism has been activated by overexpressing the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT). These animals have now been tested for their sensitivity to the polyamine analog N1,N11-diethylnorspermine (DENSPM), which is currently undergoing Phase I clinical trial. The analog is known for its ability to potently induce SSAT. Treatment for 4 days with a daily dose (125 mg/kg) of analog caused profound changes in polyamine metabolism in the transgenic animals. Liver SSAT activity was increased by approximately 800-fold while hepatic mRNA increased only 4-fold. Putrescine pools increased while spermidine and spermine pools nearly disappeared, resulting in a compensatory increase in ornithine decarboxylase activity. Similar but less profound changes were also seen in other tissues (spleen, intestine, and skin). This treatment also resulted in a 50% mortality in the transgenic animals, with no apparent histopathological changes in major organs. Nontransgenic animals exhibited no toxicity, and tissue SSAT activity was unchanged or only moderately increased. Polyamine pools were only slightly altered. Greater analog toxicity in transgenic animals may be attributable to higher tissue levels of DENSPM facilitated by SSAT-mediated decreases in spermidine and spermine. To further confirm the enhanced sensitivity of the transgenic animals to the analog, groups of nontransgenic and transgenic animals were subjected to daily injections with DENSPM. On average, transgenic mice died approximately 3 days earlier than their nontransgenic litter-mates. The findings indicate a contributing role for SSAT in whole animal toxicity by SSAT-inducing polyamine analogs.

Transpedicle screw fixation of the cervical spine.

The use of posterior cervical spine fixation has become increasingly popular in recent years. Dissatisfaction with lateral mass fixation, especially at the cervicothoracic junction, has led spine surgeons to use cervical pedicle screw fixation for reconstruction in numerous cervical spine disorders. The biomechanical advantage of a three-column fixation device implanted to secure an unstable cervical spine has proven to be a valuable tool in the spine surgeon's armamentarium. Successful placement of a pedicle screw in the cervical spine requires a sufficient three-dimensional understanding of pedicle morphology to allow accurate identification of the ideal screw axis. Variability in cadaveric based morphometric measurements used to guide the surgeon in the placement of a pedicle screw has raised legitimate concerns as to whether transpedicle fixation can be applied without significant neurovascular complications. The emergence of computer assisted image guidance systems may be implemented in the operative protocol to improve the accurate placement of a pedicle screw. The indications for placement of a pedicle screw in the cervical spine are beginning to evolve. Only surgeons experienced in transpedicle screw fixation and surgery of the cervical spine should perform this method of instrumentation.

Polyamine analogue induction of the p53-p21WAF1/CIP1-Rb pathway and G1 arrest in human melanoma cells.

Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 microM N1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16-24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.

Lysosomal sequestration of polyamine analogues in Chinese hamster ovary cells resistant to the S-adenosylmethionine decarboxylase inhibitor, CGP-48664.

CGP-48664, an inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), is presently undergoing Phase 1 clinical trials as an experimental anticancer agent. We have shown previously (D. L. Kramer et al., J. Biol. Chem., 270: 2124-2132, 1995) that Chinese hamster ovary (CHO) cells that are made resistant to the growth inhibitory effects of the drug overexpress AdoMetDC because of a stable gene amplification. Unexpectedly, these same cells (CHO/644) were found to be insensitive to the growth inhibitory effects of N1,N11-diethylnorspermine (DENSPM)-a polyamine analogue also undergoing Phase 1 clinical trials-despite accumulating approximately 5 times more analogue than parental cells. We now report that treatment of CHO/664 cells with DENSPM results in the formation of numerous large cytoplasmic vacuoles, which on the basis of electron microscopy and cytochemical staining seem to be lysosomal in origin. A series of newly established CHO cell lines made differentially resistant to 1, 3, 10, 30, and 100 microM CGP-48664 by chronic exposure were used to demonstrate that vacuole formation correlated with the accumulation of extremely high levels of DENSPM without increasing growth inhibition. These same cells were used to show that AdoMetDC gene overexpression as indicated by mRNA levels was unrelated to vacuole formation; cells resistant to 100 microM CGP-48664 displayed a 170-fold increase in AdoMetDC mRNA levels and formed vacuoles in response to DENSPM, whereas those resistant to 10 microM CGP-48664 displayed a 120-fold increase in AdoMetDC mRNA levels and failed to form vacuoles. Despite accumulating to high intracellular levels, DENSPM was much less effective than spermine at down-regulating ornithine decarboxylase and polyamine transport activities in highly resistant cells. Similarly, DENSPM was less able to induce spermidine/spermine N1-acetyltransferase activity in cells that formed vacuoles than in those that did not. Overall, natural polyamines failed to induce vacuoles and various analogues of DENSPM were used to probe the structural specificity of the effect. The data are consistent with the probability that DENSPM is sequestered to high concentrations in lysosomal vacuoles of CGP-48664-resistant cells and is, therefore, not available to interact with polyamine regulatory sites or to cytotoxically affect cell growth. In addition to implicating the lysosome as a potential new site of CGP-48664 drug action that could be involved in antitumor activity and/or host toxicities, the findings also suggest a potential mechanism of cell resistance to analogues such as DENSPM.

Developmental effect of polyamine depletion in Caenorhabditis elegans.

Ornithine decarboxylase (ODC) catalyses the conversion of ornithine to putrescine, an obligate precursor to the polyamines spermidine and spermine. We reported previously that homozygous odc-1 (pc13) worms have no detectable ODC activity. Despite their inability to make polyamines, these mutant worms appear normal, but with a slight reduction in total brood size, when grown in complex medium that presumably contains polyamines. We now show that when ODC-deficient worms are transferred to polyamine-free medium, they show a strong phenotype. odc-1 worms have two different fates, depending upon the developmental stage at which polyamines are removed. If the polyamines are removed at the L1 larval stage, the mutant animals develop into adult hermaphrodites that produce very few or no eggs. In contrast, if mutant larvae at the later L4 stage of development are transferred to polyamine-deficient medium, they develop and lay eggs normally. However, approx. 90% of the eggs yield embryos that, although well differentiated, arrest at early stage 3. Either maternal or zygotic expression of ODC provides partial rescue of embryonic lethality. Supplementing deficient medium with the polyamine spermidine allows ODC-deficient worms to develop as on complex medium. Together, these findings suggest that ODC activity is most critically required during oogenesis and embryogenesis and, furthermore, that exogenous polyamines can override the requirement for ODC activity.

Molecular cytogenetic studies of pediatric ependymomas.

Cytogenetic and molecular studies of ependymomas have previously demonstrated deletions of chromosomes 17 and 22 as frequent abnormalities, implicating inactivation of tumor suppressor genes in the pathogenesis of these tumors. In the present study, we analyzed 22 childhood ependymomas by standard cytogenetic analysis, fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR)-based microsatellite analysis of chromosomes 17 and 22. Microsatellite analysis of chromosome 6 was performed to identify submicroscopic deletions implicated by the cytogenetic studies. Among the 22 cases, we demonstrated loss of chromosome 22 in 2 patients, deletion of chromosome 17 in 2 patients, and rearrangements or deletions of chromosome 6 in 5 patients. These data do not suggest that loss of a gene on chromosome 17p plays a primary role in the initiation of pediatric ependymomas. This is in contrast to what has been reported for pediatric CNS primitive neuroectodermal tumors and malignant astrocytomas, in which deletion of 17p is regarded as a primary event. Furthermore, loss of chromosome 22 may define a subset of ependymomas more commonly seen in adults. Cytogenetic studies in this series, however, suggest that a region on the long arm of chromosome may be involved in the development and/or progression of ependymomas in children.

Human prostatic carcinoma cell lines display altered regulation of polyamine transport in response to polyamine analogs and inhibitors.

BACKGROUND: The possibility was investigated that complex homeostatic mechanisms which maintain polyamine pools in prostate-derived tumors may differ from those which are typically seen in other tissues and tumors. METHODS: Growth sensitivity and various regulatory responses were investigated in three human prostate carcinoma cell lines (LNCaP, DU145, and PC-3) treated with the inhibitor of S-adenosylmethionine decarboxylase CGP-48664 or the polyamine analog N1,N11-diethylnorspermine (DENSPM), both of which are currently undergoing phase I clinical trial. RESULTS: Prostate tumor cell lines were all similarly growth-inhibited by the inhibitor CGP-48664 (IC50 values, 1-5 microM at 72 hr), but varied considerably in their sensitivity to DENSPM. The rank-order for cell-line growth inhibition by the analog was DU145 > PC-3 > LNCaP, with IC50 values of 1, 30, and 1,000 microM, respectively. Both compounds depleted intracellular polyamine pools to levels which seemed sufficient to account for inhibition of cell growth. While polyamine enzyme regulatory responses to both CGP-48664 and DENSPM were typical of those seen in other cell types, regulation of polyamine transport differed distinctly. Based on Vmax determinations, LNCaP cells failed to upregulate transport in response to CGP-48664, while PC-3 and LNCaP cells failed to downregulate transport in response to DENSPM. CONCLUSIONS: Relative to other cell lines, polyamine transport in prostate carcinoma cell lines was found to be uniquely insensitive to regulation by polyamines or analogs. Although this did not seem to correlate with growth sensitivity to polyamine analogs in vitro, it should be therapeutically exploitable in in vivo systems.