Alkaline ceramidase 3 deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system.

Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C(18:1)-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C(18:1)-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C(18:1)-ceramide, but not C18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C(18:1)-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C(18:1)-ceramide, a potent pro-inflammatory bioactive lipid and that dysregulation of ACER3 and C(18:1)-ceramide may contribute to the pathogenesis of inflammatory diseases including cancer.

Mouse embryonic fibroblasts null for the Krüppel-like factor 4 gene are genetically unstable.

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with tumor suppressive activity in colorectal cancer. Here, we investigated whether KLF4 is involved in maintaining genetic stability in mouse embryonic fibroblasts (MEFs) isolated from mice wild type (+/+), heterozygous (+/-), or homozygous (-/-) for the Klf4 alleles. Compared to Klf4(+/+) and Klf4(+/-) MEFs, Klf4(-/-) MEFs had both a higher level of apoptosis and rate of proliferation. Quantification of chromosome numbers showed that Klf4(-/-) MEFs were aneuploid. A higher number of Klf4(-/-) MEFs exhibited gamma-H2AX foci and had higher amounts of gamma-H2AX compared to controls. Cytogenetic analysis demonstrated the presence of numerous chromosome aberrations including dicentric chromosomes, chromatid breaks, and double minute chromosomes in Klf4(-/-) cells but in few, if any, Klf4(+/+) or Klf4(+/-) MEFs. Approximately 25% of Klf4(-/-) MEFs exhibited centrosome amplification in contrast to the less than 5% of Klf4(+/+) or Klf4(+/-) MEFs. Finally, only Klf4(-/-) MEFs were capable of anchorage-independent growth. Taken together, these findings demonstrate that MEFs null for the Klf4 alleles are genetically unstable, as evidenced by the presence of aneuploidy, chromosome aberration and centrosome amplification. The results support a crucial role for KLF4 in maintaining genetic stability and as a tumor suppressor.

Ki-ras and the characteristics of mouse lung tumors.

Codon 12 mutations are frequent in the Ki-ras oncogene in human lung adenocarcinomas, but the effects of these alterations have not been well characterized in lung epithelial cells. Murine primary lung tumors derived from peripheral epithelial cells also may present Ki-ras mutations and are useful models for study of early phases of tumor development. One hypothesis is that Ki-ras mutation and/or a Ki-ras p21 increase could enhance Ki-ras p21-GTP and cell-cycle stimulation through raf-1 and extracellularly regulated protein kinases (Erks). We examined lung tumors 1-7 mm in largest dimension initiated in male Swiss mice by N-nitrosodimethylamine for pathologic type, Ki-ras mutations and levels of total Ki-ras p21, Ki-ras p21 bound to GTP, raf-1, Erk1 and Erk2 and their phosphorylated (activated) forms, and proliferating cell nuclear antigen. Total Ki-ras p21 and activated ras-GTP were not significantly greater in tumors than in normal lung or in tumors with versus those without Ki-ras mutations. Carcinomas with Ki-ras mutations were significantly smaller than those without mutations. Carcinomas were significantly larger than adenomas only for tumors without mutations. High levels of Erk2 and correlation of Erk2 amount with ras-GTP were specific characteristics of tumors with Ki-ras mutations. Size of all tumors correlated with ras-GTP but not with proliferating cell nuclear antigen. Raf-1 was expressed mainly in alveolar macrophages in normal lung but was focally upregulated in papillary areas of some tumors. The results indicate that Ki-ras influences the characteristics of lung tumors, but a linear ras-raf-Erk-cell-cycle control sequence does not adequately characterize tumorigenic events in this model. Mol. Carcinog. 28:156-167, 2000.

Oxidative DNA damage in fetal tissues after transplacental exposure to 3'-azido-3'-deoxythymidine (AZT).

The nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) has been used successfully to reduce the incidence of transplacental and perinatal transmission of the HIV virus. However, prolonged treatment with high doses of AZT is utilized in this therapy, and AZT has been found to be a perinatal carcinogen in mice. Any possible perinatal carcinogenic side effects in the human can best be managed if the mechanism is understood. AZT targets mitochondria and might cause increased intracellular production of reactive oxygen species (ROS). We tested whether transplacental AZT may cause oxidative damage in nuclear DNA of fetal tissues. CD-1 Swiss pregnant mice were treated with the transplacental carcinogenesis regimen (25 mg/day AZT, for gestation days 12-18) and tissues collected on the day of birth. Significant increases in 8-oxo-2'-deoxyguano- sine (8-oxo-dG) were found in the livers, a target tissue for transplacental carcinogenesis, and in the kidneys. A non-significant increase occurred in brain, with no change in lung. Tissues were also obtained from fetal patas monkeys (Erythrocebus patas), whose mothers had received 10 mg AZT/day during the last half of gestation. Although limited numbers of samples were available, possible increases in 8-oxo-dG were noted, relative to controls, for placenta and for fetal lung and brain (P = 0.055 for treatment-related increases in these tissues). These results suggest that an increase in reactive oxygen species could contribute to the mechanism of transplacental carcinogenesis by AZT in mice, and that this may also occur in primates.

Higher activity of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) coincides with lower background levels of 8-oxo-2'-deoxyguanosine in DNA of fetal compared with maternal mouse organs.

Mammalian homologues of Escherichia coli MutT, a protein having 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity, are thought to play the same role in preventing the incorporation of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) into DNA. One could thus expect that higher activity of 8-oxo-dGTPase should correlate with a lower background level of 8-oxo-dG in nuclear DNA. During transplacental carcinogenesis experiments, in control healthy Swiss mice on day 18 of gestation we found consistently lower levels of 8-oxo-dG in DNA in fetal livers and lungs (1.74+/-0.04 SE and 1.49+/-0.08 SE 8-oxo-dG/10(5) dG, respectively; pooled organs of fetuses of 8 dams) as compared with maternal organs (3.05+/-0.20 SE and 3.08+/-0.17 SE 8-oxo-dG/10(5) dG, respectively; n = 8). The 8-oxo-dGTPase activity determination in the same organs revealed that the lower levels of 8-oxo-dG in fetal DNA did, indeed, coincide with higher 8-oxo-dGTPase activity (48.8+/-2.6 SE and 52.5+/-2.5 SE U/mg protein in livers and lungs, respectively); and vice versa, higher 8-oxo-dG levels in DNA of maternal organs were associated with lower levels of 8-oxo-dGTPase activity (24.3+/-1.3 SE and 4.7+/-0.6 SE U/mg protein, as above). Without excluding other reasons for the relatively low 8-oxo-dG background in DNA of fetal tissues (e.g., higher level of antioxidants and antioxidative enzymes; more efficient DNA repair), this inverse relationship may support or at least does not contradict the concept of a guardian role of 8-oxo-dGTPase against 8-oxo-dGTP mutagenicity in mammalian cells.

Cadmium(II), unlike nickel(II), inhibits 8-oxo-dGTPase activity and increases 8-oxo-dG level in DNA of the rat testis, a target organ for cadmium(II) carcinogenesis.

8-Oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) is an enzyme which prevents incorporation into DNA of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) from a deoxynucleotide pool damaged by endogenous oxidants. Its inhibition may thus be carcinogenic. We previously found that Cd(II) inhibited 8-oxo-dGTPase in both cell free systems and cultured cells. To verify this finding in a relevant animal model, we investigated the effects of Cd(II) on cellular 8-oxo-dGTPase activity and nuclear DNA 8-oxo-dG levels in the rat testis, a target organ for Cd(II) carcinogenesis. Ni(II), which does not induce testicular tumors in rats and is a weaker in vitro inhibitor of 8-oxo-dGTPase than Cd(II), was investigated as a comparison. Male F344/NCr rats were given a single s.c. dose of 20 micromol Cd(II) acetate, 90 micromol Ni(II) acetate or 180 micromol sodium acetate (controls) per kg body wt and killed 2, 8, 24 or 48 h later (three rats/time point). Cd(II) caused a gradual decrease in testicular 8-oxo-dGTPase activity with time. It became significant only after 8 h post-injection (P < 0.05) and resulted in a final 50% loss of the enzyme activity at 48 h (P < 0. 01). Although the results for Ni(II) at 8 h and later were apparently lower than the controls, the decrease did not reach statistical significance. Treatment of rats with Cd(II) led to an early and progressive increase (from 130% at 2 h to 200% at 48 h versus the controls) of the 8-oxo-dG level in testicular DNA (P < 0. 05 or better). Ni(II) acetate also tended to raise the testicular 8-oxo-dG level, but the increase was transient, with an apparent maximum at 8 h, and did not approach statistical significance (P < 0. 2). Thus, Cd(II), unlike Ni(II), is able to inhibit 8-oxo-dGTPase activity and to raise 8-oxo-dG levels in rat testicular DNA. However, the time course of both effects indicates that 8-oxo-dGTPase inhibition is most likely not the sole cause of the increase in 8-oxo-dG.

[Tissue plasminogen activator in urine of patients with prostatic carcinoma and benign prostatic hyperplasia]. / Antygen tkankowego aktywatora plazminogenu w moczu chorych na raka i lagodny rozrost gruczolu krokowego.

In the 20 patients with prostatic carcinoma (PC) and the 18 with benign prostatic hyperplasia (BPH) the level of tissue type plasminogen activator (t-PA:Ag) was examined. As a compared group consisted of 24 healthy volunteers. In the urine of examined patients with PC and BPH and control the t-PA:Ag was absent or present only in trace amounts. We concluded that the t-PA:Ag in the urine of patients with PC can not be as a marker in the diagnosis of prostatic diseases especially in the prostatic carcinoma.

Construction of mobilizable cloning vectors derived from pBGS18 and their application for analysis of replicator region of a pTAV202 mini-derivative of Paracoccus versutus pTAV1 plasmid.

Two mobilizable cloning vectors, designated pABW1 and pAWB2, were constructed basing on the E. coli vector pBGS18 and oriT originating from RK2. In pABW2 the kanamycin resistance gene was replaced by a novel tetracycline resistance cassette derived from Tn1721. Both vectors, specific for E. coli, allow to perform the cloning steps in E. coli and then to efficiently transfer the constructs by conjugation to the host of choice. A vector which cannot propagate in the given host can be applied for identification of the host specific plasmid replicator regions. With the use of pABW2 we defined the minimal replicator region of pTAV202-a mini-derivative of the large pTAV1 plasmid of P. versutus. We also proved that RepC' encoded on this fragment is the principal initiator replication protein and that oriV is located along its coding sequence.

[The effect of laryngectomy on the fibrinolysis process in the blood of patients with laryngeal neoplasms]. / Wplyw laryngektomii na proces fibrynolizy we krwi chorych na raka krtani.

Some data in the literature indicate on the influence of different kind of surgery on the fibrinolytic process. Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1)--play important role in regulation of fibrinolysis. The aim of our work was to evaluate the influence of laryngectomy on fibrinolytic potential in the blood of operated patients with larynx carcinoma. Study group consisted of 20 men aged 42-75 years old with larynx carcinoma subjected to total laryngectomy. In the blood of patients we measured the concentration of t-PA:Ag, fibrinogen and FDP levels, PAI-1 activity, and ELT. The increase of t-PA and FDP concentrations, decrease of PAI-1 activity and fibrinogen level with simultaneous ELT shortening indicate on the fibrinolysis activation during the laryngectomy.

[Tissue and urokinase plasminogen activators (t-PA, u-PA) and their inhibitor PAI-1 in blood of patients with laryngeal neoplasms]. / Tkankowy i urokinazowy aktywator plazminogenu (t-PA, u-PA) oraz ich inhibitor PAI-1 we krwi chorych na raka krtani.

Plasminogen activators and inhibitors regulate a variety of physiological and pathological processes involved in tissue morphogenesis, cell differentiation, migration, cancer cell invasion and metastasis. Several reports have shown the decrease of fibrinolytic activity in the blood of cancer patients. In this study we measured the concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), activity of plasminogen activator inhibitor type 1 (PAI-1) and euglobulin lysis time (ELT) in the blood of 20 men aged 42-75 years old with planoepitheliale larynx carcinoma, and 10 healthy persons in similar age. In this study the mean values of t-PA, u-PA concentrations, PAI-1 activity and ELT in the blood of patients with larynx carcinoma were similar to the examination results of healthy persons, but 40% of our cancer patients have increased activity of PAI-1.

Effect of captopril on serotonergic mechanisms in two-kidney, one-clip renal hypertensive rats.

In 2K,1C-RHR (two-kidney, one-clip hypertensive rats) serotonergic mechanisms in blood platelets were studied. The endogenous serotonin (5-HT) concentration in whole blood and in platelets remained unchanged in relation to the sham operated rats. Also the uptake of labelled 5-HT in rats with renal hypertension was not altered. However platelets aggregability was increased in 2K,1C-RHR. Acute administration of captopril (10 mg/kg and 100 mg/kg po) diminished blood pressure but did not change either the concentration of 5-HT in whole blood and in platelets of hypertensive rats or the uptake of this amine. Platelets aggregation and the amplifying effect of 5-HT in hypertensive rats were also unchanged after acute captopril administration. Similar results were observed after its administration in a dose of 30 mg/kg for one week. Our results indicate that captopril did not affect the platelets serotonergic mechanisms in 2K,1C-RHR.