Quantitative trait loci associated with promoting effects of sodium L-ascorbate on two-stage bladder carcinogenesis in rats

In the two-stage rat bladder carcinogenesis model using N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) as an initiator and sodium L-ascorbate (SA) as a promoter, we found a notable strain difference between F344/DuCrj (F344) and WS/Shi (WS) rats in susceptibility to the promoting effect of SA. Twenty each of F344, WS and reciprocal F1 hybrid rats were given 0.05% BBN in their drinking water for 4 weeks and then a basal diet with (BBN-SA group) or without (BBN group) a 5% SA supplement for 32 weeks. In F344 and also in reciprocal F1 hybrids, the number of tumors per rat was significantly higher in the BBN-SA group than in the BBN group (P < 0.0001). In contrast, WS rats were not significantly affected by either treatment (P = 0.8). These findings indicate that F344 rats are highly susceptible to the promoter effect of SA, but WS rats are not. Linkage analysis of 108 WSx (WS x F344) F1 backcrosses revealed that this difference was related to a quantitative trait locus mapped on rat Chr. 17 (maximum LOD score, 3.86) named Bladder Tumor Susceptible-1 and possibly another locus on Chr. 5 (maximum LOD score, 2.39). This study has provided the first evidence that host genes influence the risk of bladder cancer development.     

The peroxisome proliferators are hepatocyte mitogens in chemically-defined media: glucocorticoid-induced PPAR alpha is linked to peroxisome proliferator mitogenesis

OBJECTIVES: To investigate the induction of apoptosis by methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) therapy as well as p53 alterations in a rat model of bladder cancer induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). METHODS: At 20 and 28 weeks after starting the administration of BBN, 60 rats (30 at 20 weeks, and 30 at 28 weeks) were divided into an MVAC treatment group (20 rats) and a control group of untreated rats (10 rats). After one intraperitoneal injection of MVAC, the rat bladder was obtained on day 1 or 7 for evaluation of apoptosis by biohistochemical and electron microscopic observations, and of p53 alterations immunohistochemically. RESULTS: All tumors were noninvasive transitional cell carcinoma (TCC) at 20 weeks and invasive TCC at 28 weeks. In comparison with untreated tumors, a threefold increase of apoptotic indexes (Als) was noted in invasive TCC and a twofold increase in noninvasive TCC on day 1 after MVAC therapy. Both decreased Als and a frequent occurrence of apoptotic necrosis were observed on day 7. Occurrence of tumor necrosis was not affected by MVAC therapy, and the extent of necrosis was not related to apoptosis. Detection of p53 alterations, 45% and 40% in MVAC treated and untreated tumors, respectively, did not correlate with Als. CONCLUSIONS: MVAC therapy may act through the induction of apoptosis, and invasive TCC cells may be much more sensitive to MVAC therapy in the rat model of bladder cancer. Neither spontaneous nor MVAC-induced apoptosis may be related to p53 alterations in the rat model of bladder cancer.     

Chemopreventive effects of nimesulide, a selective cyclooxygenase-2 inhibitor, on the development of rat urinary bladder carcinomas initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine

The chemopreventive potential of a selective cyclooxygenase-2 inhibitor, nimesulide (NIM), against the development of rat superficial urinary bladder carcinomas after initiation with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was examined. Six-week-old Fischer 344 male rats were given 0.05% BBN in their drinking water for 8 weeks, followed by diets supplemented with 0, 100, 200, or 400 ppm NIM for 12 weeks, and they were then sacrificed. NIM decreased, in a dose-dependent manner, the incidence of transitional cell carcinoma (TCC) to 12 of 20 (60.0%), 8 of 16 (50.0%), and 5 of 19 (26.3%) and the multiplicity of TCCs to 0.75 +/- 0.79, 0.56 +/- 0.63, and 0.37 +/- 0.78 per rat at 100, 200, and 400 ppm, respectively, as compared with the BBN alone group values of 18 of 20 (90.0%) and 2.35 +/- 1.23. NIM did not significantly affect the cell differentiation or invasiveness of TCCs. These results indicate clear chemopreventive potential of a selective cyclooxygenase-2 inhibitor against postinitiation development of superficial rat urinary bladder carcinomas.     

High susceptibility of p53(+/-) knockout mice in N-butyl-N-(4-hydroxybutyl)nitrosamine urinary bladder carcinogenesis and lack of frequent mutation in residual allele

The loss of p53 functions is considered to compromise the growth-suppression machinery of the cell and facilitate neoplastic change. In humans, genetic alteration in the p53 gene is one of the most frequently observed molecular changes in tumors, including urinary bladder carcinomas. We have investigated the susceptibility of heterozygote p53 knockout mice to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in terms of urinary bladder tumor induction. Both p53(+/-) knockout mice and C57BL/6 original parent strain were administered 0, 0.002, 0.004, 0.0075 and 0.025% BBN in the drinking water for 20 weeks. As compared with the C57BL/6 strain, greater lesion yields were observed in knockout mice after 20 weeks of treatment. Transitional cell carcinomas were found in 9 (75%) and 12 (100%) of each 12 mice of the 0.0075 and 0.025% BBN treatment groups, respectively, whereas only 1 (11%) and 6 (67%) of each 9 of the C57BL/6 mice demonstrated tumors. Preneoplastic lesions (dysplasia) were also observed more frequently in the lower dose groups in the knockout mice than C57BL/6 mice. PCR single-strand conformation polymorphism analysis followed by DNA direct sequencing of the p53 gene (exons 5-8) extracted from bladder tumors demonstrated mutations in 3 of 11 (27.3%; exon 7) and 8 of 29 (27.6%; exons 5-8) tumors in C57BL/6 and knockout mice, respectively. There was no significant difference in the mutation rates at the residual p53 gene between the two cases. All mutations observed in knockout mice were restricted to the normal allele, and none were present in the gene-targeted null allele. In a separate experiment, 5-bromo-2'-deoxyuridine labeling indices after treatment with BBN for 2 or 4 weeks were significantly higher in knockout mice than wild-type mice. Measurement of the urinary concentration of N-butyl-N-(3-carboxypropyl)nitrosamine, a proximate carcinogenic metabolite, revealed no significant differences between knockout and original parent strain after administration of 0.0075% BBN in the drinking water for 4 weeks. In conclusion, knockout mice are distinctly more sensitive to urinary bladder carcinogenesis induced by BBN than their original parent strain, as evidenced by elevated DNA synthesis during carcinogen administration and an increased tumor yield. The high susceptibility of p53 knockout mice appeared to be related to the high level of cell proliferation rather than that of N-butyl-N-(3-carboxypropyl)nitrosamine in the urine or that of mutations at the p53 gene.     

Strong promoting activity of phenylethyl isothiocyanate and benzyl isothiocyanate on urinary bladder carcinogenesis in F344 male rats

Post-initiation effects of phenylethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) on hepatocarcinogenesis and urinary bladder carcinogenesis were examined in rats pretreated with diethylnitrosamine (DEN) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Groups of 21 rats received a single intraperitoneal injection of 200 mg/kg body weight of DEN. Starting 2 days thereafter, they were administered 0.05% BBN in the drinking water for 4 weeks. Three days after completion of the carcinogen treatment, they were placed on a diet containing PEITC or BITC at a dose of 0.1%, or a basal diet alone for 32 weeks and then killed for autopsy. Further groups of 6 rats each were similarly treated with PEITC, BITC or basal diet alone for 32 weeks without prior DEN and BBN exposure. In the liver, although the incidences of liver tumors were not significantly affected, the multiplicity of foci larger than 0.5 cm in diameter was slightly increased by PEITC. In the urinary bladder, the incidences of papillary or nodular (PN) hyperplasias and carcinomas were significantly elevated by PEITC or BITC after DEN and BBN initiation. In the groups without initiation, PN hyperplasia was found in all rats of both PEITC and BITC groups, along with papillomas and carcinomas in some animals. Tumors and PN hyperplasias in the groups treated with PEITC and BITC are characterized by downward growth. Our results thus showed PEITC and BITC to be strong promoters of urinary bladder carcinogenesis with some complete carcinogenic potential.     

Osteoarthritis involving the metatarsophalangeal joints and management of metatarsophalangeal joint pain via injection therapy

We studied the effect of administration of a mixture of alanine and glutamine on the inhibition of liver regeneration caused by alcohol in rats undergoing partial hepatectomy 6 weeks after the start of alcohol administration. DNA synthesis was inhibited 24 hr after partial hepatectomy in rats given alcohol, but treatment with alanine and glutamine partially prevented this inhibition. To identify the mechanism of this effect, polyamine metabolism was studied. Administration of alcohol or alanine plus glutamine had no effect on the activity of ornithine decarboxylase, a rate-limiting enzyme of polyamine metabolism. In the liver, of the three polyamines, only the spermine concentration changed significantly. It decreased during long-term administration of alcohol, and this decrease was prevented by treatment with alanine and glutamine. The level of N(1)-acetylspermidine, the acetylated product of spermidine, was increased by alcohol, and its elevation was significantly less when alanine and glutamine were given. Hepatic spermidine/spermine N(1)-acetyltransferase, the key enzyme of polyamine acetylation, was induced by long-term administration of alcohol, and this induction was suppressed by alanine plus glutamine. The results suggest that treatment with alanine and glutamine can help to prevent the inhibition of liver regeneration caused by alcohol by maintaining the spermine level and suppressing the acetylation of spermidine.     

Chemoprevention of oral carcinogenesis by DL-alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor: dose-dependent reduction in 4-nitroquinoline 1-oxide-induced tongue neoplasms in rats

The modifying effect of three doses of DL-alpha-difluoromethylornithine (DFMO) given p.o. during the post-initiation phase of tongue carcinogenesis initiated by 4-nitroquinoline 1-oxide (4-NQO) was studied in male ACI/N rats. Animals were given 4-NQO at 20 ppm for 8 weeks in the drinking water to induce tongue neoplasms. One week after the stop of 4-NQO treatment, rats were transferred to the drinking water containing DFMO at concentrations of 100, 1000, and 2000 ppm for 25 weeks. The other groups consisted of rats given 2000 ppm DFMO alone or untreated rats. Thirty-four weeks after the start of the experiment, all animals were necropsied, and the incidences of neoplasms and preneoplastic lesions in the tongue, polyamine levels in the bloods and tongue tissues, and cell proliferation estimated by the number and area of silver-stained nucleolar organizer regions in the tongue epithelium were compared among the groups. Feeding of DFMO at all doses significantly inhibited the incidence of tongue neoplasms compared to the group given 4-NQO alone. DFMO at levels of 1000 and 2000 ppm significantly reduced the incidence of preneoplastic lesions of the tongue. Results analyzed by the linear regression method suggested a dose-dependent inhibition in the incidences of neoplastic and preneoplastic lesions of the tongue with increasing levels of DFMO. Increased levels in polyamines in the blood and tongue tissue were significantly suppressed by the treatment of DFMO. Also, silver-stained nucleolar organizer region indices were significantly reduced by the DFMO exposure. These results indicate that increasing levels of DFMO in the drinking water inhibited 4-NQO-induced tongue carcinogenesis in a dose-dependent manner and such inhibition was related to reduction in the polyamine levels of blood and tissue and decrease in the cell proliferation.     

Effect of calcium hydroxide on the dissolution of soft tissue on the root canal wall

AIMS/BACKGROUND: Given the important role of polyamines (putrescine, spermidine, spermine) in the modulation of macromolecular syntheses, gene expression and proteolysis, alterations in their metabolic pathways could be relevant during senescence. Since the few existing data address mainly polyamine biosynthesis, we studied the oxidative catabolism of polyamines in the liver of rats 3-36 months of age. METHODS: Polyamine oxidase activity was fluorimetrically measured using N1-acetylspermine as substrate. Spermidine/spermine N1-acetyltransferase and diamine oxidase were measured by radiochemical methods using labeled acetyl-coenzyme A and putrescine, respectively, as substrate. Polyamines were separated by HPLC and fluorimetrically quantified after post-column derivatization with o-phthaldialdehyde. RESULTS: Spermidine/spermine N1-acetyltransferase activity increased in 36-month-old rats and polyamine oxidase activity in 24- and 36-month-old rats. A decline in spermine and increases in spermidine and putrescine in elderly rats suggested an activation of the interconversion pathway of higher into lower polyamines. The activity of diamine oxidase, which degrades putrescine, was enhanced starting from 12 months of age. CONCLUSION: In the liver of aged rats, an increase in the catabolic enzymes leads to a reconversion of the higher polyamines to putrescine. This increased catabolism may represent an important age-related change and may contribute to impairment of the expression of growth-related genes in senescence.     

Promoting effect of monosodium aspartate, but not glycine, on renal pelvis and urinary bladder carcinogenesis in rat induced by N-butyl-N-(4-hydroxybutyl)nitrosamine

Although the incidences were relatively low, hyperplasias of the renal pelvis and the urinary bladder have been observed in Fischer-344 (F-344) rats after both sodium aspartate and glycine treatments in long-term 2-yr bioassays. In the present study, the effects of these amino acids on development of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-initiated urinary lesions were investigated in male and female F-344/DuCrj rats. F-344 rats of both sexes, 6 wk old at the commencement, were given 0.05% BBN for 4 wk and then treated with one of the amino acids at a level of 5.0% in the drinking water for the following 36 wk. Proliferative lesions in the renal pelvis often associated with necrosis and mineralization were increased in the group treated with BBN followed by sodium aspartate, but not by glycine, in both sexes. The same group demonstrated higher incidences of urinary bladder tumors with increased urinary pH and sodium concentration and decreased creatinine and uric acid, but not accompanying crystallization. These results showed a clear promoting effect of sodium aspartate for urinary carcinogenesis in rats. The mechanisms of the effect on the renal pelvis and urinary bladder might be different.     

Polyamines increase in the brain stem and cerebellum following labyrinthectomy

The goal of this investigation was to test the hypothesis that unilateral damage to the vestibular end-organ (labyrinthectomy) stimulates polyamine synthesis in central vestibular neural structures that mediate the process of behavioral recovery (vestibular compensation). Pharmacological studies have shown that compensation can be altered by alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine synthesis. Because polyamines are important in regeneration, development and modulation of N-methyl-D-aspartate (NMDA) excitatory amino acid receptors, which mediate vestibular synaptic plasticity, we investigated changes in polyamines in specific central vestibular structures after unilateral labyrinthectomy. The supernatant fraction of brain tissue homogenates was reacted with dansyl chloride. Dansylated polyamine derivatives were quantified in the vestibular nuclei, cerebellum, and inferior olive in both the control and the unilaterally labyrinthectomized guinea pig by high-performance liquid chromatography-fluorometric detection. No left-right differences in putrescine, spermidine, or spermine were detected in any brain parenchyma of controls. Polyamine imbalance, characterized by increased spermidine in the ipsilateral medial and lateral vestibular nuclei, was noted 12 and 24 h after unilateral labyrinthectomy (UL). In contrast, spermidine, spermine, and putrescine were elevated bilaterally in the cerebellum and inferior olive after UL. These biochemical changes may represent neuronal modifications to establish a balance between the vestibular nuclei after unilateral labyrinthectomy. Elucidation of the role of polyamines in central vestibular function and in vestibular compensation offers promise for the development of novel therapeutic strategies for treatment of vestibular disorders.     

Polyamine deprivation provokes an antalgic effect

It is well established that inhibition of putrescine formation using D,L-2-(difluoromethyl)ornithine and feeding a polyamine-deficient diet together with non-absorbable antibiotics (neomycin and metronidazole), prevent almost completely the growth of tumors in rats. A similar regimen given to patients with prostate cancer not only reduced the titer of prostate specific antigen in serum, but surprisingly provoked at the same time an antalgic effect. This observation led us to study the potentiation effect of polyamine deprivation on pain threshold in healthy rats. Animals were fed for 2 weeks with an artificial diet of known polyamine content, in combination with antibiotics and 2-(difluoromethyl)ornithine, and were then submitted to pain stimuli using two models, the Randall-Selitto test and the Tail-Flick test. Polyamine deprivation produced in these models an increase in the latency of the response, even under conditions which did not produce significant changes of the polyamine concentrations in blood and brain. From these observations, we may conclude that the polyamines play a role in the perception of nociceptive stimuli under physiological conditions.     

The dynamics of the imprinted H19 gene expression in the mouse model of bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine

The imprinted H19 gene product is an oncofetal RNA molecule in humans. It is expressed in fetal bladder, down-regulated postnatally and is re-expressed in human bladder carcinoma. This study was designed to investigate the dynamics of the expression of H19 in the mouse bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and its relation to stages of neoplastic transformation. BBN was administered to mice in the drinking water for 26-28 weeks. The bladders were removed at 5-10 week intervals for histopathological examination and for in situ hybridization for H19 RNA, using a 35S-labeled probe. Following BBN administration expression of H19 first appeared after 5 weeks in the lamina propria adjacent to the basement membrane, concomitant with mucosal hyperplasia. At 11 weeks focal expression was noted in epithelial cells. Invasive carcinomas, of the transitional and squamous sub-types, were seen after 20 weeks and more of BBN administration. At this stage H19 expression was observed in scattered tumor cells, in the connective tissue stroma of the tumor and in the lamina propria underlying the remaining hyperplastic/dysplastic mucosa. Abundant expression of H19 was evident in fetal bladder but was absent in normal adult bladder. We conclude that, similar to humans, the H19 gene product is an oncofetal RNA molecule in the experimental mouse model of bladder carcinoma. In this model H19 is expressed in the connective tissue of the lamina propria prior to its expression in epithelial cells, concurrent with preneoplastic changes in the transitional epithelium of the bladder.     

Polyamine changes in the vestibular nuclei of guinea pigs following labyrinthectomy

Vestibular compensation is a process of behavioral recovery from ocular, motor and postural disorders following unilateral damage to the vestibular end-organ. Although restoration of the normal resting discharge rate in the ipsilateral vestibular nuclei is important in compensation, the biochemical and molecular mechanisms mediating recovery are largely unknown. The ornithine decarboxylase polyamine pathway is activated in the nervous system following axotomy or denervation. The authors postulate that changes in polyamines mediate vestibular compensation. Within 150-micron brain stem coronal section micropunches analyzed by high performance liquid chromatography techniques, the polyamine spermidine was significantly increased in the ipsilateral lateral vestibular nucleus 8 hours following labyrinthectomy in the guinea pig model. Because naturally occurring polyamines modulate excitatory amino acid receptors (N-methyl-D-aspartate [NMDA]) which in turn mediate neurotransmission between primary afferents and second order vestibular neurons, stimulation of polyamine pathways following neural injury may play a critical role in compensation.     

Ornithine decarboxylase gene deletion mutants of Leishmania donovani

A knockout strain of Leishmania donovani lacking both ornithine decarboxylase (ODC) alleles has been created by targeted gene replacement. Growth of Deltaodc cells in polyamine-deficient medium resulted in a rapid and profound depletion of cellular putrescine pools, although levels of spermidine were relatively unaffected. Concentrations of trypanothione, a spermidine conjugate, were also reduced, whereas glutathione concentrations were augmented. The Deltaodc L. donovani exhibited an auxotrophy for polyamines that could be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplementation restored intracellular pools of both putrescine and spermidine, exogenous spermidine was not converted back to putrescine, indicating that spermidine alone is sufficient to meet the polyamine requirement, and that L. donovani does not express the enzymatic machinery for polyamine degradation. The lack of a polyamine catabolic pathway in intact parasites was confirmed radiometrically. In addition, the Deltaodc strain could grow in medium supplemented with either 1,3-diaminopropane or 1, 5-diaminopentane (cadaverine), but polyamine auxotrophy could not be overcome by other aliphatic diamines or spermine. These data establish genetically that ODC is an essential gene in L. donovani, define the polyamine requirements of the parasite, and reveal the absence of a polyamine-degradative pathway.     

Chemopreventive efficacy of piroxicam administered alone or in combination with lycopene and beta-carotene on the development of rat urinary bladder carcinoma after N-butyl-N-(4-hydroxybutyl)nitrosamine treatment

The effects of the non-steroidal anti-inflammatory drug (NSAID) piroxicam and the carotenoids lycopene and beta-carotene, alone or in combination, on the development of rat superficial urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were studied. Male Fischer 344 rats, 6 weeks old, were given 0.05% BBN in the drinking water for 8 weeks followed by administration of piroxicam (0.0075% in the diet), lycopene (0.0025% in the drinking water) and/or beta-carotene (0.0025% in the drinking water) for 12 weeks, then killed for histological analysis of urinary bladder lesions. Cell proliferation potential was analyzed by immunohistochemical staining of the proliferative cell nuclear antigen (PCNA). Piroxicam alone, piroxicam+lycopene, and piroxicam +lycopene+ beta-carotene all significantly decreased the incidences and numbers of transitional cell carcinomas (TCCs), but the combination of piroxicam with carotenoids did not result in a clear improvement in the preventive potential of piroxicam. Piroxicam+ beta-carotene also caused a significant reduction and lycopene alone a slight but not significant reduction in the number of TCCs. In contrast, beta-carotene alone and lycopene+ beta-carotene were without inhibitory influence on any of the lesion categories examined, and the latter significantly increased the proportion of high-grade TCCs. Nevertheless, all of the chemopreventive agents, either alone or in combination, significantly decreased the TCC PCNA index, the effect extending to the surrounding epithelium in the piroxicam+lycopene and piroxicam+lycopene+beta-carotene groups. These results indicate that the NSAID piroxicam may be a more effective chemopreventive agent than lycopene and beta-carotene for superficial urinary bladder carcinogenesis.     

Human erythrocyte polyamine levels after partial hepatectomy

BACKGROUND/AIM: There are various indices of liver regeneration, but no clinically useful index that reflects the current status of liver regeneration. We assayed human erythrocyte polyamine levels after partial hepatectomy to define the relationship between erythrocyte polyamine levels and liver regeneration. MATERIALS AND METHODS: Levels of human erythrocyte polyamines (putrescine, spermidine, and spermine) were assayed by high-pressure liquid chromatography in 91 patients after partial hepatectomy and in 13 patients after surgery other than partial hepatectomy (controls). Of the patients after partial hepatectomy, 37 underwent hepatectomy of 20% or more of the liver (group A), 27 underwent segmentectomy or subsegmentectomy of the liver amounting to less than 20% of the liver (group B), and 27 underwent an operation smaller in scale than sub-segmentectomy (group C). RESULTS: The greater the proportion of the liver resected, the greater was the percent increase. In groups A, B, and C, erythrocyte levels of spermidine and spermine increased after surgery compared with the base line, and were significantly higher at 7 or 14 days, decreasing later. The differences in spermidine among the three groups were significant. CONCLUSIONS: After partial hepatectomy, the erythrocyte polyamine levels, especially the level of spermidine, were related to the proportion of liver resected. They seemed to reflect the degree of liver regeneration.     

A histopathological mapping study of the urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in dogs

Bladder tumors were induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in five Beagles and four mongrel dogs. The tumors were observed for long periods and the tumor progression was traced using histopathological mapping. The results indicated (1) that low-dose BBN over a long period induced multiple low-grade (G1-2) and low-stage (pTa-1) papillary tumors, resembling superficial bladder cancer in humans; (2) that high-dose BBN over a short period induced high-grade (G2-3) and high-stage (pT3b) nonpapillary tumors and carcinoma in situ (CIS) resembling invasive cancer and CIS in humans; (3) that beagle dogs required longer periods and higher total doses of BBN as compared with mongrel dogs; (4) that the tumors induced by low-dose BBN in beagles were observed without BBN as long as the animals lived, and neither increasing numbers of tumors nor malignant features such as deep infiltration and metastasis was observed; and (5) that low-dose BBN seems to induce mild dysplasia, which is followed by Brunn's nest-like proliferation in the lamina propria and nodular change, eventually leading to the development of papillary noninvasive transitional cell carcinoma (TCC); and that high-dose BBN seems to induce severe dysplasia which leads to CIS and nonpapillary invasive TCC. These results may contribute to clarifying the natural history of human bladder cancer.     

Polyamine analogue induction of programmed cell death in human lung tumor cells

The naturally occurring polyamines putrescine, spermidine, and spermine are required for cell growth. Based on this requirement, several polyamine analogues that interfere with polyamine function and metabolism have been synthesized as antineoplastic agents. The symmetrically substituted N1,N12-bis(ethyl)spermine (BESpm), and unsymmetrically substituted N1-ethyl-N11-[(cyclopropyl)methyl]-4, 8-diazaundecane (CPENSpm) have previously been shown to cause rapid cytotoxicity of NCI H157 cells, with concurrent high induction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. However, the precise mechanism(s) of the cytotoxic action of the compounds is not known. We now demonstrate that treatment with either BESpm or CPENSpm results in morphological and biochemical changes consistent with the activation of programmed cell death pathways, and that the unsymmetrically substituted CPENSpm more rapidly activates the death program. These studies suggest that the cell type-specific cytotoxicity of these polyamine analogues may be a result of their ability to selectively activate the cell death pathway in sensitive phenotypes and indicate that the relationship between the structure of the polyamine analogues and the ability to induce programmed cell death should be investigated.     

Kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth

The kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth were studied. The steady deceleration of the specific growth rate with increasing tumor mass that was observed was attributable to a prolongation of the cell cycle, particularly of the S and G2 phases. The cell cycle time (Tc) was 43.3 hr (TG1 equals 10.8, TS equals 26.8, and TG2 equals 5.7 hr) on the seventh day of growth and 76.0 hr (TG1 equals 14.0, TS equals 52.0, and TG2 equals 10.0 hr) on the tenth day of growth. The growth fraction showed a decrease from 0.77 to 0.60 during the 7- to 10-day tumor growth interval. The cell death rate remained low and essentially unchanged during this period. A high correlation was found between polyamine synthesis (ornithine decarboxylase activity) and the specific growth rate; the correlation coefficient was 0.985. There was also a high positive correlation between the cellular polyamine (spermidine and spermine) and nucleic acid content (spermidine: DNA equals 0.916, spermine: DNA equals 0.947, spermidine:RNA equals 0.907, and spermine: RNA equals 0.881). These observations suggest that there may be a functional coupling between polyamines and nucleic acids, and they support the hypothesis that polyamines play an important role in DNA replication and cell division.     

Inhibition by dehydroepiandrosterone of butylated hydroxyanisole (BHA) promotion of rat-bladder carcinogenesis and enhancement of BHA-induced forestomach hyperplasia

The effects of dehydroepiandrosterone (DHEA) with/without ribonucleoside (RNs) supplementation on butylated hydroxyanisole (BHA) bladder-tumor promotion and forestomach carcinogenesis were investigated. Male F344 rats were given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for 4 weeks and then received basal diet or diet containing BHA, DHEA, a mixture of RNs, BHA + DHEA or BHA + DHEA + RNs for 32 weeks. The occurrences of papillomas and carcinomas in the urinary bladder were increased in the groups given BHA or BHA + DHEA + RNs, as compared with control group values. In comparison with the BHA group, the BHA + DHEA group incidences and numbers of these tumors were decreased. However, the incidence and multiplicity of papillomas in the group given BHA + DHEA + RNs were again elevated. DNA synthesis levels in normal-appearing bladder epithelium, but not tumor cells, were closely correlated with the observed level of promotion in most groups. The case of DHEA alone proved exceptional in that DNA synthesis was markedly decreased without any significant influence on lesion development. In the forestomach, DHEA, which itself was associated with slight although non-significant hyperplasia, enhanced BHA-induced epithelial lesions, characterized by marked basal-cell proliferation and keratin-cyst formation, independently of additional RNs administration. Our results suggest that the anti-promoting effects of DHEA in the bladder depend on a deficiency in the pentose phosphates necessary for production of nucleosides. Organ-specific modulation is indicated by the enhancing effects of DHEA on BHA-induced forestomach hyperplasia.