Cyclic AMP-mediated suppression of normal and neoplastic B cell proliferation is associated with regulation of myc and Ha-ras protooncogenes

Cyclic AMP functions as a negative regulator of cell proliferation in a variety of cell systems. We show here that the proliferation of normal and neoplastic B cells can be inhibited by high intracellular levels of cAMP. Thus forskolin treatment of the neoplastic B precursor cell line Reh induced a rapid increase in the cAMP level, which was followed by an accumulation of cells in the G0/G1 phase of the cell cycle over a period of 2-3 days. Similar inhibition of Reh cell proliferation after 3 days was observed whether forskolin was present continuously or only during the first 5 hr. Both c-myc and c-Ha-ras protein levels were transiently down-regulated at 4 hr of forskolin treatment, suggesting that these protooncogenes play a role in the process leading to cAMP-mediated growth cessation. Northern-blot analysis showed that the steady-state levels of c-myc RNA rapidly declined in all phases of the cell cycle, to return to control levels within a time period of 24 hr. In contrast, the c-Ha-ras mRNA level was steadily maintained. Thus the expression of c-myc and c-Ha-ras protein was regulated at different metabolic levels. The reduced proliferative capacity of the B precursor cell line in the presence of forskolin was not linked to induced differentiation. This was judged from the lack of appearance of three different B cell differentiation markers; cytoplasmic immunoglobulin heavy chain and two antigens recognized by the monoclonal antibodies B1 (CD20) and HH1 (CD37). We also showed that forskolin partially inhibited the proliferation of normal B lymphocytes stimulated by anti-immunoglobulins (anti-mu) and B cell growth factor (BCGF). The burst of c-myc mRNA during activation of normal B cells was also reduced by forskolin.     

Expression of the cytosolic and particulate forms of transglutaminase during chemically induced rat liver carcinogenesis

Transglutaminase (EC 2.3.2.13) activity in chemically induced rat hepatocellular carcinomas was reduced by some 65% when compared to normal rat livers. The majority of the remaining activity (approx. 85%) was found in the particulate fraction. The use of non-ionic detergent to extract the transglutaminase activity present in both normal and tumour tissue followed by its separation on a Mono-Q column revealed two distinct peaks of activity. These peaks of activity were equivalent to those previously identified as a membrane-bound transglutaminase and the more characteristic cytosolic or tissue transglutaminase. The ratio of the activity of the cytosolic enzyme to that of the membrane-bound enzyme in normal liver was calculated as 5:1. In hepatocellular carcinomas, this ratio was reduced to 0.4:1. No significant change in the activity of the membrane-bound enzyme was detectable in tumour tissue. Comparison of the cytosolic enzyme found in hepatocellular carcinomas with that found in normal liver indicated no change in its molecular weight, Km,app for putrescine incorporation into N,N'-dimethylcasein and sensitivity to activation by Ca2+. These observations suggest that the reduction in transglutaminase activity observed in the hepatocellular carcinoma is due to a selective reduction in the expression of the cytosolic transglutaminase.     

Acute oxidative stress is associated with cell proliferation in the mouse liver

Oxidative stress is known to produce tissue injury and to activate various signaling pathways. To investigate the molecular events linked to acute oxidative stress in mouse liver, we injected a toxic dose of paraquat. Liver necrosis was first observed, followed by histological marks of cell proliferation. Concomitantly, activation of the MAP kinase pathway and increased levels of the anti-apoptotic protein Bcl-XL were observed. Gene expression profiles revealed that the differentially expressed genes were potentially involved in cell proliferation. These data suggest that paraquat-induced acute oxidative stress triggers the activation of regeneration-related events in the liver.     

Heat induced liver cell proliferation in the livebearing fish Poeciliopsis

Synopsis Liver regeneration is induced by heat stress in the small viviparous fish, Poeciliopsis. Acute exposure to sublethal temperatures, one to two degrees below their killing temperature, damages tissue and initiates liver cell proliferation in P. lucida, P. monacha, and P. monacha-lucida hybrid clones, SYN-4 and SYN-5. Regeneration of liver cells began within 1–2 days following heat stress and proceeded over 5 days. Peak cell proliferation occurred 2–3 days after treatment in fish of all four genotypes. Cell proliferation was induced in the two all-female clones, SYN-4 and SYN-5, by exposure to 40.5° C for 60 minutes. This treatment imposed mortalities of 17.9% and 16.7%, respectively, whereas reduction of the temperature to 39.5°C and reduction of the time to 30 minutes resulted in no mortalities without significantly lowering the level of cell proliferation (p > 0.05). Liver cell proliferation induced by both heat treatments was significantly higher (p < 0.05) in the SYN-5 hybrids than in SYN-4. The induction of liver cell proliferation with sublethal temperature exposures is discussed as it may relate to chemical carcinogenesis in both feral and laboratory fish. Acute heat exposure may be used experimentally in fish as an independent stimulus for liver cell proliferation in carcinogenesis studies. In poikilothermic animals-heat exposure offers an alternative to surgical removal of approximately two-thirds of the liver, the method most frequently used in rodents to study the process of liver regeneration.     

天然和氧化修饰型LDL、VLDL及HDL对培养人动脉平滑肌细胞原癌基因fos及myc表达的影响

动脉平滑肌细胞（ＳＭＣ）是动脉粥样硬化（ＡＳ）斑块中的主要细胞,它的增殖在ＡＳ形成过程中极其重要。脂蛋白和氧化修饰型脂蛋白对ＳＭＣ增殖的影响以及ＳＭＣ增殖与原癌基因异常表达的关系是当前ＡＳ发病机制研究的热点之一。我们在建立人主动脉ＳＭＣ体外培养方法的基础上,观察了ＬＤＬ,ＶＬＤＬ及ＨＤＬ和相应的氧化修饰型脂蛋白对培养人ＳＭＣｆｏｓ,ｍｙｃ,ｅｒｂ－Ｂ原癌基因转录表达的影响。结果表明：①ＨＤＬ对ＳＭＣｆｏｓ,ｍｙｃ基因表达无影响;②ＬＤＬ和ＶＬＤＬ有使这些基因表达增加的趋势,但与对照比较差异不显著（Ｐ＞０．０５）;③ＯＸ－ＶＬＤＬ,ＯＸ－ＶＬＤＬ和ＯＸ－ＨＤＬ有使ＳＭＣｆｏｓ,ｍｙｃ基因表达显著增强的作用（Ｐ＜０．０１）,且其作用较相应的天然脂蛋白大（Ｐ＜０．０１）．上述结果说明：ＬＤＬ,ＶＬＤＬ,ＯＸ－ＬＤＬ,ＯＸ－ＶＬＤＬ和０Ｘ－ＨＤＬ的致ＡＳ作用可能与刺激ＳＭＣｆｏｓ和ｍｙｃ癌基因表达增加有关。     

N-Nitrosomorpholine induced alterations of unscheduled DNA synthesis, mitochondrial DNA synthesis and cell proliferation in different cell types of liver, kidney, and urogenital organs in the rat

In order to measure rates of unscheduled DNA synthesis (UDS), mitochondrial DNA synthesis, and cell proliferation, i.e. factors relevant in the early phase of carcinogenesis, young rats received by gavage 200 mg/kg N-nitrosomorpholine (NNM) or vehicle (distilled water), and were injected with 3H-thymidine 24 h later. Autoradiographs from liver, kidney, urethra, prostate, seminal vesicle, and ductus deferens were prepared from deparaffinized sections, using a 250-day exposure time. In the liver, UDS was at least doubled in 2n and 4n hepatocytes. Approximately 3% of these hepatocytes exhibited a fourfold increase in UDS. Such strongly labeled cells were only observed in the liver following NNM exposure. With the exception of renal epithelial cells of the proximal tubule, UDS in epithelial cells of bladder, urethra, ductus deferens, seminal vesicle and prostate was decreased in NNM-exposed rats. Mitochondrial DNA synthesis and cell proliferation were significantly increased only in hepatocytes, and were decreased in all other monitored organs in NNM-exposed rats. The strongly increased UDS and more moderately increased mitochondrial DNA synthesis in a subgroup of hepatocytes suggest that possibly some unrepaired damage persists in the DNA of these cells. The latter cells may be the precursors of so-called foci of hepatocellular alteration, which appear later during the process of carcinogenesis. The increased UDS but decreased rate of proliferation in the renal proximal tubule cells might be related to renal carcinogenesis which is observed in NNM-exposed rats after a long latency period.     

Expression of TTK, a novel human protein kinase, is associated with cell proliferation.

We have isolated the full-length sequence for a unique human kinase, designated TTK. TTK was initially identified by screening of a T cell expression library with anti-phosphotyrosine antibodies. The kinases most closely related to TTK are the SPK1 serine, threonine and tyrosine kinase, the Pim1, PBS2, and CDC2 serine/threonine kinases, and the TIK kinase which was also identified through screening of an expression library with anti-phosphotyrosine antibodies. However, the relationships are distant with less than 25% identity. Nevertheless, TTK is highly conserved throughout phylogeny with hybridizing sequences being detected in mammals, fish, and yeast. TTK mRNA is present at relatively high levels in testis and thymus, tissues which contain a large number of proliferating cells, but is not detected in most other benign tissues. Freshly isolated cells from most malignant tumors assessed expressed TTK mRNA. As well, all rapidly proliferating cell lines tested expressed TTK mRNA. Escherichia coli expressing the complete kinase domain of TTK contain markedly elevated levels of phosphoserine and phosphothreonine as well as slightly increased levels of phosphotyrosine. Taken together, these findings suggest that expression of TTK, a previously unidentified member of the family of kinases which can phosphorylate serine, threonine, and tyrosine hydroxyamino acids, is associated with cell proliferation.     

碱性成纤维细胞生长因子对大鼠肾小球系膜细胞原癌基因c-fos和c-myc表达的影响

在建立大鼠肾小球系膜细胞（ＭＣ）体外培养方法的基础上,通过３Ｈ－ＴｄＲ参入实验,ＲＮＡ印迹分析和斑点杂交观察ｂＦＧＦ对ＭＣＤＮＡ合成及原癌基因ｃ－ｆｏｓ和ｃ－ｍｙｃ表达的影响．结果表明,ｂＦＧＦ作用于ＭＣ１８ｈ,ＭＣ的３Ｈ－ＴｄＲ参入率明显增加（Ｐ＜０．０５）,２４ｈ达到高峰（Ｐ＜０．０１）;ｂＦＧＦ显著诱导原癌基因ｃ－ｆｏｓ和ｃ－ｍｙｃ表达,其表达活性分别于３０ｍｉｎ和１ｈ达到高峰．提示ｂＦＧＦ是ＭＣ的强效丝裂原,其对ＭＣＤＮＡ合成的促进作用与诱导原癌基因ｃ－ｆｏｓ和ｃ－ｍｙｃ表达有关．     

Expression of the Ha-ras suppressor family member 5 gene in the maturing rat testis

We analyzed the gene expression of Ha-ras suppressor family member 5 (Hrasls5), which is considered to modulate the Ha-ras signaling cascade, from maturing rat testis. Expression was detected primarily in the spermatocytes in the maturing rat testis. The Hrasls5 gene product might function as a tumor suppressor as well as in spermatogenesis, as deduced from its amino acid sequence.     

Stimulating effect of pentagastrin on cancer cell proliferation kinetics in chemically induced colon cancer in rats

Pharmacological doses of pentagastrin or gastrin are known to stimulate cell proliferation in normal colonic epithelium but the growth-promoting effect of gastrin on colon carcinoma is still controversial. In this study morphological parameters were measured to study the effect of pentagastrin (240 micrograms/kg) on the cell proliferation kinetics in experimental tumours. Colon cancer was produced in rats by weekly injections (20 mg/kg b.wt.) of 1.2-dimethylhydrazine for 24 weeks. Tritiated thymidine was given after administration of pentagastrin or the control solution to the animals. 75% of the animals from the pentagastrin group and 66% of the controls had at least one colon cancer. Autoradiographs of the colonic tumors were performed and the percentage of labeled cells in the cancer cell population was determined after counting 4000 to 16,000 cancer cells per tumor. The labeling index for cancer cells in the pentagastrin-treated group (21.49 +/- 1.76%) was higher (P less than 0.01) than in the control group (14.76 +/- 0.66%). In a second study vincristine sulphate (1 mg/kg) was given to the animals 20 h after administering pentagastrin or the control solution. The percentage of arrested metaphases in the tumours was determined after counting 10,000 to 24,000 cancer cells per histological section. Pentagastrin increased (P less than 0.01) the mean metaphase index by 108% (4.9 +/- 0.44% vs 2.35 +/- 0.32%). These data indicate that cell cycle manipulation of colon cancer is possible with hormonal peptides.     

Abnormal intranuclear and cytoplasmic formations associated with a chemically induced,transplantable chicken sarcoma

Thirty GRCH/15 tumors (a 1, 2, 5, 6-dibenzanthracene-induced chicken sarcoma) were examined in the light and the electron microscope. Associated with the sarcoma were two types of abnormal intranuclear lesions, one in the form of a vacuole, the other as an aggregate containing glycogen. In the electron microscope, one type of lesion observed showed an organized microfibrillar structure. Abnormal cytoplasmic formations occurred as massed clusters of thread-like or tubular material, which gave rise to small bodies with concentric shell structure; similar bodies were found associated with vacuoles.     

Partial purification and immunoreactivity of an 80000-molecular-weight polypeptide associated with peroxisome proliferation in rat liver

Hypolipidaemic drugs and industrial plasticizers such as di-(2-ethylhexyl) phthalate, which cause proliferation of hepatic peroxisomes, also cause an increase in an 80000-mol.wt. polypeptide in the liver of rats and mice. This polypeptide has been designated as PPA-80 (PPA, for peroxisome-proliferation-associated; 80 for 80000mol.wt.). The polypeptide PPA-80 was purified to over 90% purity from livers of rats treated with the peroxisome proliferators Wy-14,643, nafenopin, tibric acid and clofibrate by a single-step preparative sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic procedure. The antibodies raised against the PPA-80 polypeptide isolated from livers of rats treated with Wy-14,643 cross-reacted with polypeptide PPA-80 purified from the livers of rats treated with Wy-14,643, as well as from the livers of rats treated with nafenopin, tibric acid and clofibrate. The anti-(polypeptide PPA-80) antibodies did not cross-react with catalase, a marker enzyme for peroxisomes, or with NADPH–cytochrome P-450 reductase, which has the same approximate mol.wt., 80000. The intensity of immunoprecipitin bands formed with microsomal, large-particle and postnuclear fractions from livers of animals pretreated with peroxisome proliferators was significantly greater compared with equal amounts of protein from corresponding fractions obtained from control animals, suggesting that these agents all enhance the synthesis of the same 80000-mol.wt. polypeptide. Although the polypeptide PPA-80 was increased in the postnuclear, large-particle and microsomal fractions of livers of rats pretreated with peroxisome proliferators, the relative abundance of this peptide in the peroxisome-rich light-mitochondrial fraction and its lack in highly purified mitochondrial fractions suggest the localization of this polypeptide in peroxisomes and/or microsomal fraction. Additional studies are needed to establish unequivocally the subcellular localization of the polypeptide PPA-80 and to ascertain if this polypeptide is identical with the multi-functional protein displaying enoyl-CoA hydratase and β-hydroxyacyl-CoA dehydrogenase activities that was purified by Osumi & Hashimoto [(1979) Biochem. Biophys. Res. Commun. 89, 580–584].     

Expression of the rat prothymosin alpha gene during T-lymphocyte proliferation and liver regeneration.

Prothymosin alpha (ProT alpha) is a widely distributed acidic protein whose function has been related to cell proliferation. We have analyzed the expression of the rat ProT alpha gene in several proliferative systems: concanavalin A (ConA)/interleukin-2-stimulated thymocytes, ConA-stimulated splenic T-lymphocytes, and hepatocytes proliferating during liver regeneration. In these systems, ProT alpha mRNA was detected in all stages of the cell cycle, with maximal increments (2-4-fold) at the beginning of the S phase. By contrast, the mRNAs for proliferating cell nuclear antigen/cyclin and histone H3, two cell-cycle-regulated proteins, were hardly detected in resting cells but increased notably at the G1/S boundary and in the S phase, respectively. Treatment of T-cells with the calcium ionophore A23187 increased ProT alpha mRNA levels 2.5-fold, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, had no effect on ProT alpha gene expression. Incubation of ConA-stimulated T-cells with hydroxyurea, a DNA synthesis inhibitor, did not decrease the levels of ProT alpha mRNA, indicating that its expression is independent of DNA synthesis. These findings suggest that ProT alpha is required throughout all the stages of the cell cycle, resembling a constitutively expressed gene rather than one strictly involved in cell proliferation.