Assessment of NMDA receptor activation in vivo by Fos induction after challenge with the direct NMDA agonist (tetrazol-5-yl)glycine: effects of clozapine and haloperidol.

Induction of Fos protein by the potent and direct NMDA agonist (tetrazol-5-yl)glycine (TZG) was examined in mice. Effects of antipsychotic drugs were assessed on this in vivo index of NMDA receptor activation. TZG induced the expression of Fos in a neuroanatomically selective manner, with the hippocampal formation showing the most robust response. In mice genetically altered to express low levels of the NR1 subunit of the NMDA receptor, TZG-induced Fos was reduced markedly in comparison to the wild type controls. TZG-induced Fos was also blocked by the selective NMDA antagonist MK-801. Pretreatment of mice with clozapine (3 and 10 mg/kg) reduced TZG-induced Fos in the hippocampal formation but not in other brain regions. Haloperidol at a dose of 0.5 mg/kg did not antagonize TZG induced Fos in any region. Haloperidol at a dose of 1.0 mg/kg did attenuate the induction of Fos by TZG in the hippocampus but not in other brain regions. The relatively high dose (1 mg/kg) of haloperidol required to block effects of TZG suggests that this action may not be related to the D(2) dopamine receptor-blocking properties, since maximal D(2) receptor blockade was probably achieved by the 0.5 mg/kg dose of haloperidol. The antidepressant drug imipramine (10 or 20 mg/kg) did not antagonize TZG induced Fos in any brain region. The data suggest that clozapine can reduce excessive activation of NMDA receptors by TZG administration in vivo at doses relevant to the drugs' actions in rodent models of antipsychotic activity. Whether or not this action of clozapine contributes to its therapeutic properties will require further study.

Brain oxygen metabolism may relate to the temperature gradient between the jugular vein and pulmonary artery after cardiopulmonary resuscitation.

OBJECTIVE: A gradient between the jugular vein temperature and core body temperature has been reported in animal and clinical studies; however, the pathophysiological meaning of this phenomenon remains unclear. This study was conducted to identify the temperature gradient between the jugular vein and pulmonary artery in comatose patients after cardiopulmonary resuscitation. MATERIALS AND METHODS: The temperatures of the jugular vein and pulmonary artery were measured in 19 patients at 6 and 24 hours after cardiopulmonary resuscitation. Jugular venous blood saturation (SjO2; %) was also measured concomitantly. The patients were divided into 2 groups: high SjO2 (SjO2 > 75%: H-group; n = 10) and normal SjO2 (SjO2 < or = 75%: N-group; n = 9). The temperature gradient was calculated by subtracting the temperature of the pulmonary artery from that of the jugular vein (jugular - pulmonary = dT degrees C). Statistical significance was defined as p < 0.05. RESULTS: dT was significantly lower in the H-group than in the N-group at 6 hours (0.120 +/- 0.011: mean +/- SD vs. 0.389 +/- 0.036: p = 0.0012) and 24 hours (0.090 +/- 0.005 vs. 0.256 +/- 0.030: p = 0.0136) after cardiopulmonary resuscitation. CONCLUSION: The temperature gradient between the jugular vein and pulmonary artery was significantly lower in patients with high SjO2 after cardiopulmonary resuscitation. This temperature gradient may be reflected in brain oxygen metabolism.

Abundant expression of immunoreactive endothelin 1 in mammary phyllodes tumor: possible paracrine role of endothelin 1 in the growth of stromal cells in phyllodes tumor.

Immunoreactive endothelin 1 (irET-1) concentrations were measured in extracts prepared from 4 phyllodes tumors and 14 fibroadenomas. irET-1 was detectable in all tissue extracts by specific radioimmunoassay, and the mean concentration of irET-1 was 18-fold and 27-fold higher in tissue extracts from phyllodes tumors than in those from intracanalicular fibroadenomas and pericanalicular fibroadenomas, respectively. Reverse-phase high-performance liquid chromatography coupled with radioimmunoassay in the extracts from phyllodes tumors revealed one major irET-1 component corresponding to human standard ET-1. Furthermore, immunocytochemical staining for ET-1 revealed that numerous ET-1-immunoreactive cells were seen in the epithelial cells but not in the stromal cells, suggesting that ET-1 is synthesized by the epithelial component of phyllodes tumors. A possible paracrine role of ET-1 in the growth of this rare tumor which is characterized by its prominent stromal cellularity is discussed.

Eradication diminishes enhancing effects of Helicobacter pylori infection on glandular stomach carcinogenesis in Mongolian gerbils.

To investigate the nature of the link between Helicobacter pylori (Hp) infection and stomach carcinogenesis, a study of the glandular stomach of Mongolian gerbils (MGs) was performed. MGs were treated with N-methyl-N-nitrosourea (MNU), followed by inoculation with Hp (groups 1 and 2) or without Hp (group 3), or infected with Hp (groups 4 and 5) or inoculation without Hp (group 6) followed by MNU administration. At week 21, the animals in groups 2 and 5 underwent an eradication procedure. At week 50, the incidences of adenocarcinomas in group 1 (15 of 23) and group 4 (9 of 26) were significantly higher than in group 3 (1 of 15) and group 6 (1 of 18), respectively. Moreover, those in group 2 (5 of 24) and group 5 (2 of 22) were lower than in groups 1 and 4, respectively. This study shows that Hp eradication may be useful as a prevention approach against stomach cancer.

Interleukin 6 stimulates the production of immunoreactive endothelin 1 in human breast cancer cells.

To investigate the potential regulation of endothelin 1 production in human breast cancer cells, we measured the release of immunoreactive endothelin 1 (ir-ET-1) from the MCF-7 and ZR-75-1 breast cancer cell lines in response to various agents including estrogen and tamoxifen as well as several cytokines. ir-ET-1 was detected in conditioned medium of MCF-7 cells and ZR-75-1 cells by specific radioimmunoassay. Among the agents tested, estrogen, tamoxifen, tumor necrosis factor, gamma-interferon, interleukin (IL) 1, and transforming growth factor beta had no effect on ir-ET-1 secretion by these breast cancer cells. However, IL-6 (20 ng/ml) treatment of MCF-7 cells and ZR-75-1 cells caused maximal increases in the amount of ir-ET-1 secreted into the culture medium to 206 and 314% of basal values after 6 h, respectively. This effect of IL-6 on ir-ET-1 secretion was inhibited by actinomycin D and cycloheximide, indicating that IL-6 stimulates de novo synthesis of ir-ET-1 at a transcriptional level. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay in the conditioned medium from IL-6-treated cells revealed one major ir-ET-1 component corresponding to human standard ET-1. The present study demonstrates the potential for IL-6 to stimulate ir-ET-1 production in human breast cancer cells, which may participate in the process of acute phase reactant-like expression of this peptide and/or in the process of IL-6 enhanced breast cancer cell motility, the latter being recently clarified.

The clp1 gene of the mushroom Coprinus cinereus is essential for A-regulated sexual development.

Sexual development in the mushroom Coprinus cinereus is under the control of the A and B mating-type loci, both of which must be different for a compatible, dikaryotic mycelium to form between two parents. The A genes, encoding proteins with homeodomain motifs, regulate conjugate division of the two nuclei from each mating partner and promote the formation of clamp connections. The latter are hyphal configurations required for the maintenance of the nuclear status in the dikaryotic phase of basidiomycetes. The B genes encode pheromones and pheromone receptors. They regulate the cellular fusions that complete clamp connections during growth, as well as the nuclear migration required for dikaryosis. The AmutBmut strain (326) of C. cinereus, in which both A- and B-regulated pathways are constitutively activated by mutations, produces, without mating, dikaryon-like, fertile hyphae with clamp connections. In this study we isolated and characterized clampless1-1 (clp1-1), a mutation that blocks clamp formation, an essential step in A-regulated sexual development, in the AmutBmut background. A genomic DNA fragment that rescues the clp1-1 mutation was identified by transformations. Sequencing of the genomic DNA, together with RACE experiments, identified an ORF interrupted by one intron, encoding a novel protein of 365 amino acids. The clp1-1 mutant allele carries a deletion of four nucleotides, which is predicted to cause elimination of codon 128 and frameshifts thereafter. The clp1 transcript was normally detected only in the presence of the A protein heterodimer formed when homokaryons with compatible A genes were mated. Forced expression of clp1 by promoter replacements induced clamp development without the need for a compatible A gene combination. These results indicate that expression of clp1 is necessary and sufficient for induction of the A-regulated pathway that leads to clamp development.

Vascular endothelial growth factor and platelet-derived endothelial cell growth factor are frequently coexpressed in highly vascularized human breast cancer.

Vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) are known to be angiogenic growth factors in vitro and in vivo. In this study, we have investigated the relationship between VEGF expression and PD-ECGF expression in human breast cancer tissues using immunocytochemical methods. Of 152 primary breast cancers, 84 (55.3%) and 71 (46.7%) were positive for VEGF and PD-ECGF, respectively. Fifty-three (63. 1%) of 84 VEGF-positive tumors had a PD-ECGF-positive phenotype, whereas only 18 (26.5%) of 68 VEGF-negative tumors had a PD-ECGF-positive phenotype. There was a significant correlation between the VEGF expression and PD-ECGF expression (P < 0.01). As a single factor, VEGF expression and PD-ECGF expression were significantly associated with an increase in the microvessel density assessed by the immunocytochemical analysis using antifactor VIII-related antigen mAb. Interestingly, in addition, of 53 tumors with more than 100 microvessel counts/1 mm2, 40 (75.5%) had both VEGF- and PD-ECGF-positive phenotypes. It was found that VEGF and PD-ECGF were frequently coexpressed in highly vascularized tumors with high microvessel counts. It is suggested that VEGF and PD-ECGF might cooperatively function in the neovascularization of human breast cancer.

Serum concentrations of hepatocyte growth factor in breast cancer patients.

Hepatocyte growth factor (HGF) was first identified as a potent stimulator of hepatocyte growth and DNA synthesis. Later, it was shown that HGF can promote cell motility and cell proliferation in various types of cells, including tumor cells and endothelial cells. We have examined serum concentrations of HGF in breast cancer patients using an ELISA. Of 134 primary breast cancer patients, 49 (36.6%) showed a significant increase in the circulating level of HGF as compared to healthy controls. The increase in the HGF level was significantly associated with axillary lymph node metastases and histological evidence of venous invasion. No significant correlation between serum HGF concentrations and intratumoral HGF concentrations was found; however, the removal of the primary tumor clearly decreased the serum HGF level, suggesting that the elevation of HGF in the serum was tumor related. Twenty-nine (82.9%) of 35 patients with recurrent breast cancer had an increase in the serum HGF level. The HGF level was significantly higher in patients with liver metastases compared to those with other sites of metastases. Postoperative sequential examinations confirmed that the increase in the serum HGF level was associated with the appearance of relapse. In conclusion, the serum HGF level was significantly increased in breast cancer patients. Circulating HGF might play important roles in tumor progression in this malignancy.

Environmental radioactivity of water samples collected in Higashi-Hiroshima campus, Hiroshima University, Japan.

The relation between concentration of elements and microbial activity in the water samples of Higashi-Hiroshima Campus, Hiroshima University was investigated. Energy dispersive X-ray spectroscopy revealed that microbial mat contains iron, aluminium, silicon and phosphorus. Model experiment revealed that the potassium was adsorbed by living microorganism in the microbial mats, while it was not adsorbed by dead microbial mat. Iron was adsorbed by both living and dead microbial mats. The present results explain the increase in the total ß-radioactivity of water sample in summer and the decrease in winter.

Elevated plasma levels of interleukin-1 receptor antagonist and interleukin-10 in patients with acute myocardial infarction.

The study was undertaken to measure plasma interleukin-1 (IL-1) receptor antagonist and IL-10 concentrations in patients with acute myocardial infarction and to analyze their relationship to the hemodynamics, severity, and prognosis of myocardial infarction in its acute stages. We attempted to define the kinetics of IL-1 receptor antagonist and IL-10 in patients with acute myocardial infarction (n = 34, age 42-91 years, mean 68 years). Plasma IL-1 receptor antagonist and IL-10 levels were measured by enzyme-linked immunosorbent assay. Patients in group A (n = 17) had uncomplicated acute myocardial infarction (Killip class I). Patients in group B (n = 17) had severe acute myocardial infarction (Killip class II, III, or IV). Peak Il-1 receptor antagonist and IL-10 levels in group B were significantly higher (p < 0.05) than those of group A. In group B, the peak IL-1 receptor antagonist levels were significantly correlated with white blood cell counts (r = 0.63, p = 0.006), pulmonary capillary wedge pressure (r = 0.78, p = 0.0002), and cardiac index (r = -0.51, p = 0.04). Peak IL-10 levels were significantly correlated with white blood cell counts (r = 0.60, p = 0.01), the pulmonary wedge pressure (r = 0.73, p = 0.0008), and cardiac index (r = -0.50, p = 0.04). Moreover, a significant correlation was found between the peak IL-1 receptor antagonist and IL-10 levels (r = 0.91, p < 0.0001). The peak IL-1 receptor antagonist levels in nonsurvivors (n = 13) were significantly higher (p < 0.01) than those in survivors (n = 21). The plasma IL-1 receptor antagonist and IL-10 levels were closely correlated with the severity of hemodynamics in acute myocardial infarction and with the clinical status of patients with severe acute myocardial infarction. Results suggest that plasma IL-1 receptor antagonist and IL-10 can serve as prognostic indicators in cases of sever acute myocardial infarction.

Polymorphisms of two fucosyltransferase genes (Lewis and Secretor genes) involving type I Lewis antigens are associated with the presence of anti-Helicobacter pylori IgG antibody.

Helicobacter pylori attach to the gastric mucosa with adhesin, which binds to Lewis b (Le(b)) or H type I carbohydrate structures. The Secretor (Se) gene and Lewis (Le) gene are involved in type I Le antigen synthesis. The present study was performed to investigate the possibility that Se and Le gene polymorphisms alter the risk of H. pylori infection. Two hundred thirty-nine participants were genotyped for Se and Le and tested for the presence of anti-H. pylori IgG antibodies. Using the normal gastric mucosa from 60 gastric cancer patients, we assessed immunohistochemically whether type I Le antigen expression depended on the Se and Le genotypes. The H. pylori infection rate was positively associated with the number of Se alleles (se/se group, 45.1%; Se/se group, 64.6%; and Se/Se group, 73.3%) and negatively associated with the number of Le alleles (le/le group, 76.4%; Le/le group, 68.3%; and Le/Le group, 55.6%). When the subjects were classified into three groups [low risk, (se/se, Le/Le) genotype; high risk, (Se/Se, le/le), (Se/Se, Le/le), and (Se/se, le/le) genotypes; moderate risk, other than low- or high-risk group], the odds ratio relative to the low-risk group was 3.30 (95% confidence interval, 1.40-7.78) for the moderate-risk group and 10.33 (95% confidence interval, 3.16-33.8) for the high-risk group. Immunohistochemical analysis supported the finding that Se and Le genotypes affected the expression of H. pylori adhesin ligands. We conclude that Se and Le genotypes affect susceptibility to H. pylori infection.

Age-related increase in nitric oxide synthase activity in senescence accelerated mouse brain and the effect of long-term administration of superoxide radical scavenger.

The levels of nitric oxide (NO) and NO synthase (NOS) activities were compared in the brains of young adult (3 months old), aged (11 months old) and TJ-960 administered (11 months old) senescence accelerated mice (SAM), of which the SAMP8 substrain is inferior in acquisition of learning due to the abnormality of glutamatergic neurotransmission in the cerebral cortex. TJ-960, which is based on the Kampo (Japanese traditional herbal medicine) prescription Sho-saiko-to-go-keishi-ka-shakuyaku-to, acts as a superoxide radical scavenger and attenuates the deterioration of neuronal activity associated with aging. We administered TJ-960 orally for 5 months. In the cerebral cortex of aged SAMP8, NOS activity was increased compared with that of young adult SAMP8. Though TJ-960 did not alter the contents of NO in any brain region compared with those in aged SAMP8, it did prevent the increase in NOS activity in the aged cerebral cortex. Our data suggest that NOS activity may increase to compensate for the reduced sensitivity of the NO reaction system in the aging process, and that TJ-960 may normalize this increased NOS activity in the cerebral cortex, although further work is clearly needed to ascertain maintenance in the acquisition of learning.

Intravascular malignant lymphomatosis manifesting clinically as bilateral sudden hearing loss and cytomegalovirus encephalitis.

A 49-year-old man developed bilateral sudden hearing loss followed by cerebral infarction and cytomegalovirus (CMV) encephalitis. Autopsy confirmed intravascular malignant lymphomatosis (IML). A literature review indicates that hearing loss can be the initial manifestation of IML and also that CMV infection is more than an opportunistic infection and may participate in the cognitive manifestations in IML.

Structure-activity relationships of arginine analogues on nitric oxide synthase activity in the rat brain.

Nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from L-arginine (Arg) which has a guanidino group in its molecule. We examined the effect of 23 different Arg analogues on NOS activity in the rat brain. Though homoarginine, epsilon-guanidinocaproic acid and canavanine act as substrates of NOS, production of NO from them was lower than that from Arg. alpha-Guanidinoglutaric acid (2-GGA) and arcaine inhibited NOS activity at levels equal to NG-monomethyl-L-arginine (MeArg), a well known NOS inhibitor. Though almost all previously reported NOS inhibitors were synthesized by substituting the guanidino nitrogen of Arg, the guanidino nitrogens of arcaine and 2-GGA were not substituted. Furthermore, 2-GGA is a known endogenous convulsant in mammals, and arcaine, which was isolated from a marine mollusc, is also a convulsive substance. Hence, 2-GGA and arcaine will be excellent drugs to investigate not only the chemical nature of NOS but also the physiologic function of NO.

Ameliorative effect of NC-1900, a new AVP4-9 analog, through vasopressin V1A receptor on scopolamine-induced impairments of spatial memory in the eight-arm radial maze.

The mechanism by which NC-1900, a new pGlu-Asn-Cys(Cys)-Pro-Arg-Gly-NH(2) (AVP(4-9)) analog, improves spatial memory in rats using an eight-arm radial maze was examined. Even at very low doses (0.2 ng/kg for s.c., 1 microg/kg for p.o., 1 fg for i.c.v.) NC-1900 improved scopolamine-induced impairment of spatial memory. NC-1900 (1 ng/kg, s.c.) also improved impairment of spatial memory induced by pirenzepine, a muscarinic(1) (M(1)) receptor antagonist, and by KN-62, a Ca2+/calmodulin (CaM)-dependent protein kinase II inhibitor. [Pmp(1), Tyr(Me)(2)]-Arg(8)-vasopressin, a vasopressin(1A) (V(1A)) receptor antagonist, and nicardipine, L-type Ca2+ blocker, but not OPC-31260, a V(2) antagonist, suppressed the effect of NC-1900 on scopolamine-induced impairment of spatial memory. A microdialysis study showed that NC-1900 did not affect acetylcholine release in the ventral hippocampus (VH) of intact rats or of scopolamine-treated rats. NC-1900 (1 microM) increased [Ca2+](i) in the VH than in the dorsal hippocampus (DH). Pretreatment with nicardipine (1 microM) and Ca2+ -free conditions inhibited the NC-1900-induced [Ca2+](i) response in the VH. Whereas co-administration of NC-1900 (1 microM) and carbachol (500 microM) increased [Ca2+](i) in the VH. Moreover, nicardipine concentration-dependently inhibited the increase in [Ca2+](i) induced by the co-administration of NC-1900 and carbachol in the VH. These results suggest that NC-1900 activates the V(1A) receptor at the postsynaptic cholinergic nerve, and causes a transient influx of intracellular Ca2+ through L-type Ca2+ channels, to interact with the M(1) receptor. The activation of these Ca2+ -dependent processes induced by NC-1900 may be involved in the positive effect of NC-1900 on scopolamine-induced impairment of spatial memory.

Summation of initiation activities of low doses of the non-hepatocarcinogen 1,2-dimethylhydrazine in the liver after carbon tetrachloride administration.

Summation of initiation by low doses of the indirect-acting non-hepatocarcinogen, 1,2-dimethylhydrazine (DMH) after proliferative stimulation with a necrogenic dose of carbon tetrachloride (CCl4) was investigated in terms of the induction of glutathione S-transferase placental form (GST-P) positive liver cell foci. Cell kinetics of liver after CCl4 i.g. treatment were examined with bromodeoxyuridine (BrdU) labeling (experiment I). To assess the correlation between cell proliferation and induction of liver cell foci, DMH (10 mg/kg i.g.) was administrated to 7-week-old male F344 rats at 12, 24, 36, 48, 60, 96 h after CCl4 i.g. and initiated populations expanded using the resistant hepatocyte model (experiment IIA). Subsequently, effects of repeated administration (10 mg/kg, four times, i.g.) of DMH were compared with the results of a single administration (40 mg/ kg, i.g.) with the same total dose (experiment IIB). In experiments I and IIA, the numbers and areas of GST-P-positive foci increased with the BrdU labeling index at the time of DMH treatment (maximum after 60 h). In experiment HB, repeated exposure of DMH at 10 mg/kg, four times resulted in significant (P<0.05) increase in number and area of GST-P-positive foci compared with the single administration (40 mg/kg). Thus, multiple low dose treatments during cell proliferation might be most effective for detection of weak initiation activity.

Induction of invasive squamous cell carcinomas in the forestomach of (C3H x MSM)F1, MSM, and C3H mice by N-methyl-N-nitrosourea and mutational analysis of the H-ras and p53 genes.

Genetic analysis of tumors developing in F1 hybrids between genetically separate strains of mice makes it possible to search for loss of heterozygosity (LOH), information on which provides clues to finding tumor-suppressor genes. For this purpose, however, reproducible carcinogenic conditions for the organ of interest need to be first determined. In the present study, a forestomach model of squamous cell carcinomas (SCCs), induced in (C3H x MSM)F1 mice by N-methyl-N-nitrosourea (MNU), was established and mutational changes in the H-ras and p53 genes were examined in tumors. Male (C3H x MSM)F1, MSM and C3H mice were given MNU by i.g. intubation once a week at a dose of 0.03 mg/g body weight for 10 weeks, then kept without further treatment. At experimental weeks 38-46, markedly invasive SCCs were observed in the forestomach at incidences of 9/14 (64.3%), 9/16 (56.3%), and 2/10 (20.0%), respectively. In the three strains of mice, DNA analysis of SCCs by PCR-SSCP analysis followed by direct DNA sequencing revealed low incidences of point mutations in the H-ras (4/20, 20%) and p53 (3/20, 15%) genes. The results demonstrate the usefulness of the present animal experimental protocol for induction of high grade SCC in the forestomach of (C3H x MSM)F1 mice, and suggest the possibility that point mutations in the H-ras or p53 genes may play some role in pathways leading to the development of such lesions.

Effects of carbon tetrachloride administration on initiation of liver cell foci by the non-hepatocarcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene (B(a)P).

In a development trial for an initiation bioassay system, cell proliferation kinetics after partial hepatectomy (PH) or CCl4 administration (1 ml/kg b.w., i.g.) and the effect of administration time after PH or CCl4 treatment on liver cell foci induction by the direct and indirect non-hepatocarcinogens, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene (B(a)P) were investigated. Male F344 rats were killed 12, 18, 24, 36, 48, 72 or 96 h after PH or CCl4 treatment and liver cell proliferation was examined with the bromodeoxiuridine (BrdU) labeling method. Appreciable increase in the BrdU labeling index was observed 18-36 h after PH with a peak at 24 h, and 18-72 h following treatment with CCl4 with a peak at 48 h. MNNG (80 mg/kg i.g.) or B(a)P (100 mg/kg i.g.) were administered to 7-week-old male F344 rats at various times after PH or CCl4 treatment and lesion induction was assessed using the resistant hepatocyte model. MNNG caused significant numbers of glutathione S-transferase placental form (GST-P)-positive liver cell foci in rats when given 12-36 h after PH, with a peak at 24 h. In contrast, the numbers of foci induced by B(a)P were maximal with exposure at 12 h after PH. In the CCl4 study, both MNNG and B(a)P induced significant increase in GST-P-positive liver cell foci when given 12-72 h after CCl4 treatment, with a peak at 48 h, the results being directly in line with the changes in BrdU labeling. From these findings, it is concluded that initiation assay protocols with a CCl4 proliferative stimulus to hepatocytes may prolong the appropriate administration period for effective detection of the initiation potential of both direct and indirect carcinogens targeting sites other than the liver.

p53 mutations in transitional cell carcinomas of the urinary bladder in rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine.

Involvement of p53 gene alterations has been demonstrated in a variety of human neoplasias including urinary bladder carcinomas. N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis models in rodents have been widely used to study carcinogenic processes in this organ. In the present study, transitional cell carcinomas induced in the urinary bladders of male F344 rats treated with 0.05% BBN for 16 or 32 weeks and then sacrificed at experimental week 32 were analyzed for mutational changes in the p53 and H-ras genes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and subsequent DNA sequencing. The total p53 mutation incidences were 3/10 (30%) and 8/12 (66.7%) in rats treated with BBN for 16 weeks followed by 16 weeks' non-treatment, or in rats treated with BBN for 32 weeks, respectively, while the H-ras mutation incidences were 0/10 (0%), and 1/12 (8.3%), respectively. The present results indicate that mutations in the p53 gene might be involved in the process of urinary bladder carcinogenesis by BBN as part of a multistep pathway. However, considering the decreasing tendency in lesions with p53 mutations after stopping BBN administration, a p53 mutation alone would not appear to be sufficient to give a marked selective advantage to mutant cells. No evidence of H-ras mutation involvement was gained even for the late course of rat urinary bladder carcinogenesis.

Chemosensitivity of micrometastases and circulating tumor cells to uracil and tegafur as evaluated using LacZ gene-tagged Lewis lung carcinoma cell.

The chemosensitivity of the sequence of steps responsible for metastasis formation including circulating tumor cells and micrometastases to a 5-fluorouracil derivative (UFT) was examined with a novel micrometastasis model featuring Lewis lung carcinoma cells tagged with the bacterial LacZ gene and hygromycinR gene (hygR). Metastases in the lung could be specifically detected at the single-cell level by X-Gal staining after inoculation of LacZ-transfected tumor cells. Spontaneous metastasis in mice bearing subcutaneous primary tumors was significantly inhibited by UFT at doses of 15-20 mg/kg when it was orally administered from day 14, during the early stage of micrometastasis formation, but not by late administration from day 22. Experimental pulmonary metastasis was also inhibited without significant toxic side effects by oral administration of UFT at a daily dose of 20 mg/kg from day 4, when the tumor cells start new growth in the lung, but not by daily treatment from 8 days after intravenous injection. Oral administration of UFT had no effect on tumor cell arrest in the lung as detected by X-Gal staining. Furthermore, PCR analysis revealed that circulating tumor cells in the peripheral blood of mice bearing primary tumors after subcutaneous injection of hygR-tagged tumor cells were significantly reduced by the oral administration of UFT for 7 days in a dose-dependent manner. These results indicate that circulating tumor cells in the peripheral blood and micrometastases in the initial growth phase in the lungs are sensitive to this chemotherapeutic agent, and suggest that micrometastasis formation can be effectively inhibited by long-term oral administration of anticancer agents with minimal toxic side effects.