Acquired resistance to temsirolimus in human renal cell carcinoma cells is mediated by the constitutive activation of signal transduction pathways through mTORC2.

BACKGROUND: The objective of this study was to characterise the mechanism underlying acquired resistance to temsirolimus, an inhibitor of mammalian target of rapamycin (mTOR), in renal cell carcinoma (RCC). METHODS: A parental human RCC cell line, ACHN (ACHN/P), was continuously exposed to increasing doses of up to 20 µM of temsirolimus, and a cell line resistant to temsirolimus (ACHN/R), showing a sixfold higher IC50 than that of ACHN/P, was developed. RESULTS: Following treatment with temsirolimus, phosphorylation of S6 kinase in ACHN/P was markedly inhibited, whereas there was no detectable expression of phosphorylated S6 in ACHN/R before and after temsirolimus treatment. However, AKT and p44/42 mitogen-activated protein kinase (MAPK) were constitutively phosphorylated even after temsirolimus treatment in ACHN/R, but not in ACHN/P. There was no significant difference between the sensitivities of ACHN/P and ACHN/R to KU0063794, a dual inhibitor of mTOR complex 1 (mTORC1) and mTORC2. Similar sensitivities to temsirolimus in ACHN/P and ACHN/R could be achieved by additional treatment with specific inhibitors of AKT- and MAPK-signaling pathways. CONCLUSION: The activation of signal transduction pathways via mTORC2, but not via mTORC1, may have an important role in the acquisition of a resistant phenotype to temsirolimus in RCC.

Enhanced progression of human prostate cancer PC3 cells induced by the microenvironment of the seminal vesicle.

The objective of this study was to characterise the mechanism mediating the prostate cancer progression induced by the microenvironment of seminal vesicle (SV). The invasive potential of PC3 cells significantly increased after treatment with extract from SV of NOD/SCID mouse. Among several growth factors and cytokines that were present in the SV extract, transforming growth factor-beta(1) (TGF-beta(1)) significantly enhanced the invasive potential of PC3 cells; however, the additional treatment with neutralising antibody against TGF-beta(1) suppressed the enhanced invasive potential induced by the SV extract. Changes in the invasive potential in PC3 cells after treatment with the SV extract and/or TGF-beta(1) were in proportion to those in the production of urokinase-type plasminogen activator (uPA) by PC3 cells. Tumour growth as well as the incidence of lymph node metastasis in NOD/SCID mice after the injection of PC3 cells into the SV were significantly greater than those after the injection into the prostate. These findings suggest that the microenvironment of SV enhances the progression of prostate cancer through a stimulated invasive potential, and that enhanced uPA production in prostate cancer cells induced by TGF-beta(1) could therefore be one of the most important mechanisms involved in the progression of prostate cancer after SV invasion.

Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 microM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca(2+) ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca(2+) was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca(2+).

The relationship between calcium, MAP kinase, and DNA synthesis in the sea urchin egg at fertilization.

Fertilization releases the brake on the cell cycle and the egg completes meiosis and enters into S phase of the mitotic cell cycle. The MAP kinase pathway has been implicated in this process, but the precise role of MAP kinase in meiosis and the first mitotic cell cycle remains unknown and may differ according to species. Unlike the eggs of most animals, sea urchin eggs have completed meiosis prior to fertilization and are arrested at the pronuclear stage. Using both phosphorylation-state-specific antibodies and a MAP kinase activity assay, we observe that MAP kinase is phosphorylated and active in unfertilized sea urchin eggs and then dephosphorylated and inactivated by 15 min postinsemination. Further, Ca(2+) was both sufficient and necessary for this MAP kinase inactivation. Treatment of eggs with the Ca(2+) ionophore A23187 caused MAP kinase inactivation and triggered DNA synthesis. When the rise in intracellular Ca(2+) was inhibited by injection of a chelator, BAPTA or EGTA, the activity of MAP kinase remained high. Finally, inhibition of the MAP kinase signaling pathway by the specific MEK inhibitor PD98059 triggered DNA synthesis in unfertilized eggs. Thus, whenever MAP kinase activity is retained, DNA synthesis is inhibited while inactivation of MAP kinase correlates with initiation of DNA synthesis.

The 25 degrees-36 degrees region of the Bacillus subtilis chromosome: determination of the sequence of a 146 kb segment and identification of 113 genes.

We determined a 146 kb contiguous sequence at the 25 degrees-36 degrees region of the Bacillus subtilis chromosome containing the amyE-srfA segment. Among the 113 ORFs identified, 33 are already known. functions were assigned to 38 ORFs by a search of non-redundant protein sequence data banks and those of 16 ORFs were suggested through significant similarity with reported sequences. The amino acid sequences of 13 of the ORfs were similar to proteins of unknown function of Escherichia coli, Haemophilus influenzae and other species. We did not find similarities for 29 ORFs to any known proteins. The 146 kb region is rich in enzymes (35 ORFs) related to the metabolism of low molecular mass compounds and five genes for surfactin production occupy about 26 kb of the region.

Oxygen-controlled regulation of the flavohemoglobin gene in Bacillus subtilis.

A gene, hmp, which encodes a ubiquitous protein homologous to hemoglobin was isolated among genes from Bacillus subtilis that are induced under anaerobic conditions. The hmp protein belongs to the family of two-domain flavohemoproteins, homologs of which have been isolated from various organisms such as Escherichia coli, Alcaligenes eutrophus, and Saccharomyces cerevisiae. These proteins consist of an amino-terminal hemoglobin domain and a carboxy-terminal redox active site domain with potential binding sites for NAD(P)H and flavin adenine dinucleotide. The expression of hmp is strongly induced upon oxygen limitation, and the induction is dependent on a two-component regulatory pair, ResD and ResE, an anaerobic regulator, FNR, and respiratory nitrate reductase, NarGHJI. The requirement of FNR and NarGHJI for hmp expression is completely bypassed by the addition of nitrite in the culture medium, indicating that fnr is required for transcriptional activation of narGHJI, which produces nitrite, leading to induction of hmp expression. In contrast, induction of hmp was still dependent on resDE in the presence of nitrite. A defect in hmp in B. subtilis has no significant effect on anaerobic growth.

Substrate specificity of bovine liver cytosolic neutral alpha-mannosidase activated by Co2+.

A cytosolic neutral alpha-mannosidase was purified from bovine liver. Its molecular weight was found to be 500,000 on gel filtration. The activity of the enzyme toward Man alpha 1-6-(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc-PA was increased 26-fold by preincubation with 1 mM Co2+. Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc was hydrolyzed by the enzyme to Man alpha 1-3Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc, which was further hydrolyzed to Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc. The rate of hydrolysis was 15-fold greater than that of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. This substrate specificity suggested that the enzyme could be involved in the degradation of oligomannose-type sugar chains with one GlcNAc residue released from glycoproteins by endo-beta-N-acetylglucosaminidase, and supported a pathway for glycoprotein catabolism via oligomannosyl glycans with one GlcNAc residue proposed on the basis of an earlier study on a cytosolic neutral alpha-mannosidase from Japanese quail oviduct [Oku, H. and Hase, S. (1991) J. Biochem. 110, 982-989].

DNA-dependent protein kinase inhibitor (OK-1035) suppresses p21 expression in HCT116 cells containing wild-type p53 induced by adriamycin.

The tumor growth suppressor p21 has been shown to be induced by wild-type p53 (wt-p53) and to be a potent inhibitor of cyclin-dependent kinases and PCNA/DNA polymerase delta. Although wt-p53 is reported to be phosphorylated by several protein kinases, the function and significance of the phosphorylation of wt-p53 are not yet fully understood. Using OK-1035, a selective inhibitor of DNA-dependent protein kinase (DNA-PK), we demonstrated the importance of the phosphorylation of wt-p53 by DNA-PK in the DNA damage-mediated expression of the p21 gene. Treatment of HCT116, a human colon carcinoma cell line, with adriamycin induced the expression of wt-p53 and p21. By addition of OK-1035 to this culture, the induction of p21 protein was significantly decreased in a dose-dependent manner, whereas wt-p53 induction was not affected. Northern blot analysis revealed that suppression of p21 protein expression by OK-1035 resulted from reduction in the level of p21 mRNA. OK-1035 did not directly affect the binding ability of wt-p53 to its consensus DNA sequence. Our observations support the idea that wt-p53 induces the transcriptional activation of the p21 gene only after it is phosphorylated by DNA-PK.

A research to develop a predicting system of mammalian subacute toxicity. I. Prediction of subacute toxicity using the biological parameters of acute toxicities.

Predicting equations of subacute toxicity were developed by analyzing rat acute and subacute toxicity data of 56 chemicals of various structures. Minimum or 10% effect level in acute or subacute toxicity was estimated as a "biological parameter". Good regression equations were established between the geometrical means ("combined parameters") of any two of the parameters of acute and subacute toxicities and introduction of log P to the equations improved the correlations with a statistically significant multiple regression coefficient. The lowest predicted effect level of the subacute toxicity, which is selected from the data calculated by the above several correlations, can predict the upper limit of the no observed effect level. In recent years, for research and development of new chemical substances, it becomes one of the important factors that these have a lower environmental load in nature. Eventually, it becomes essential to evaluate not only their acute effects on human or environments, but also their chronic influences when they are to be exposed for a long period of time, and the cost for such verification is becoming a breaking factor for research and development. Thus, the development of a new technique which estimates the environmental load of a chemical substance including toxic effects on human with lower cost is now being attempted. For example, the development of in vitro new alternative methods using cultured cells, the utilization of a data base or software which relates mammalian and environmental toxicities and so on are internationally carried forwards. As for the latter, quantitative structure-activity relationship (QSAR) techniques have been applied practically to decide appropriate toxicity tests needed for regulatory purposes by EPA/TSCA, ITC/TSCA and FDA/FDAA in the USA. In addition, it was concluded recently in a joint meeting of EU and US-EPA that the approach by QSAR techniques was useful to specify the new chemical substances which are to be required toxicological examinations. In these QSAR techniques, however, while there are some considerations about common mechanisms of toxicity among chemicals with a similar structure (Congeneric chemicals, Congeners) for establishing of correlation formulas, for chemicals with various structures (Non-congeneric chemicals, Non-congeners) there often lack such common considerations. In addition, biological or physiological factors which are basic toxic indices are often ignored. In a previous study, we researched acute and subacute oral toxicities of industrial common chemicals in rats and reported the followings; 1) their subacute toxicological spectrum in target organs/tissues and morphologic changes was very limited and specific, 2) the important targets were liver, kidneys, blood (spleen) and stomach and these are considered the sites of dominant exposure due to the kinetics of chemical substances, 3) the morphological changes were hepatocellular hypertrophy, deposition of substance in renal tubules, extramedullary hematopoiesis in spleen and mucous lesion in stomach, and these are implying adaptation such as induction of drug-metabolizing enzymes, overload to renal function, anemia from erythrocyte destruction, and direct reaction, respectively, 4) there seems to occur a series of direct and adaptive reactions to exclude the "foreign compounds" which do not show any specific biological activities, 5) it is also considered that there is a possibility to establish a correlation between toxicological findings or target organs/tissues of both acute and subacute toxicities by their continuity. Therefore, in the present study, a predicting equation of subacute (28-day repeated dose) toxicity is attempted to develop from acute (single dose) toxicity data by considering both common mechanisms and biological factors for non-congeneric industrial chemical substances.

OK-1035, a selective inhibitor of DNA-dependent protein kinase.

Screening for inhibitors of DNA-dependent protein kinase (DNA-PK) revealed 3-cyano-5-(4-pyridyl)-6-hydrazonomethyl-2-pyridone, designated OK-1035, to be a potent and selective inhibitor. When a synthetic peptide was used as a substrate, OK-1035 caused 50% inhibition of DNA-PK activity at 8 microM, a concentration more than 50 times lower than those required against seven other protein kinases tested. OK-1035 inhibited the phosphorylation by DNA-PK of consensus peptide as well as that of recombinant human wild type-p53. Kinetic studies indicated that OK-1035 inhibited DNA-PK activity in an ATP-competitive manner.

Stimulation of SV40 DNA replication by the human c-myc enhancer.

In earlier studies we had shown that a transcriptional enhancer sequence exists about 2 kb upstream of the human c-myc gene. The core sequence necessary for enhancer activity was defined therein as a 21 bp nucleotide element, which also showed autonomous replicating activity [EMBO J. (1988) 7, 3135-3142; EMBO J. (1989) 8, 4273-4279]. Recently, several reports have substantiated the notion that transcription and replication can be concertedly regulated in a larger number of cases than expected. In this report, we took the simian virus 40 (SV 40) ori/promoter as a model system. The SV40 enhancer is known to enhance transcription from its ori/promoter, but to reduce its replication (probably due to a negative feedback). The SV40 enhancer was replaced by the c-myc enhancer core in order to see its effect upon SV40 DNA replication and transcription. The results showed that besides stimulating transcription, the c-myc enhancer promoted SV40 DNA replication in monkey CosI cells. Stimulation was only observed when the c-myc enhancer was inserted in the 'up-to-down' orientation to the SV40 promoter. The promoting function of the c-myc enhancer on DNA replication correlated with the transcriptional activation function, as determined by systematic point mutations introduced within the 21 bp core sequence.

[MRI of lymphedema using short-TI-IR (STIR)].

Thirty four cases with lymphedema of the extremities were examined with MRI at 0.5 tesla. On T1-weighted image, the enlarged subcutaneous tissue and the subcutaneous trabecular structures were seen in all cases. Moreover, the trabecular structures in the enlarged subcutaneous tissue showed low signal intensity on T1-weighted image and high signal intensity on T2-weighted image in all cases. Additionally, in 12 of 15 cases examined by Short-TI-IR (STIR) image, the trabecular structures and fluid collections in the subcutaneous tissue were shown more definitely in high signal intensity than by T2-weighted image. We consider MRI using STIR is to be useful in the evaluation of edematous disease.

[Clinical comparison of 99mTc-diethylenetriaminepentaacetic acid-human serum albumin (99mTc-HSA-D) and 99mTc-human serum albumin (99mTc-HSA) for cardiac blood pool imaging].

99mTc-HSA-D has been developed as a new blood pool scanning agent. Clinical comparison of 99mTc-HSA-D and 99mTc-HSA was made in 16 cases. The activity concentration of 99mTc in blood was measured during 2 hours after the injection in five cases. 99mTc-HSA-D showed higher concentration compared to 99mTc-HSA with the passage of time. Quantitative analysis of contrast between left ventricle and septum was performed on end diastolic frames of gated images 10 minutes after the injection. There was no obvious difference between 99mTc-HSA-D and 99mTc-HSA. The subjective comparison of detectability of lesions between the two agents was performed on three directional gated images. 99mTc-HSA-D was superior to 99mTc-HSA, because the images of the latter deteriorated with the passage of time. On anterior view images 1 hour after the injection, left ventricle/lung and abdominal aorta/background count ratios were greater for 99mTc-HSA-D in many cases. There was no obvious difference in liver/background and kidney/background count ratios between the two agents. Urinary excretion of 99mTc was considerably lesser for 99mTc-HSA-D. The results indicated that 99mTc-HSA-D was superior to 99mTc-HSA for cardiac blood pool imaging.