Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells,with Limited Impact on Cancer Hallmark Functions

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca²⁺ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na⁺. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.     

EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart

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M(en)TORship lessons on life and death by the integrated stress response

Cells employ pro-survival and pro-adaptive pathways to cope with different forms of environmental stress. When stress is excessive, and the damage caused by it is unsustainable, cells engage pro-death pathways, which are in place to protect the host from the deleterious effects of harmed cells. Two important pathways that determine the balance between survival and death of stressed cells are the integrated stress response (ISR) and the mammalian target of rapamycin (mTOR), both of which converge at the level of mRNA translation. The two pathways have established avenues of communication to control their activity and determine the fate of stressed cells in a context-dependent manner. The functional interplay between the ISR and mTOR may have significant ramifications in the development and treatment of human diseases such as diabetes, neurodegeneration and cancer.     

Aim‐then‐shoot anemotaxis involved in the hopping approach of potato tuberworm moth Phthorimaea operculella toward a sex pheromone source

Male moths locate conspecific females by pheromone‐induced upwind flight maintained by detecting a visual flow, termed optomotor anemotaxis. Their behavioural pattern is characterized by an upwind surge in response to a pheromone stimulus and crosswind casting after odour loss, which is considered to be reset and restarted on receipt of another pheromone pulse. However, pheromone‐stimulated males of the potato tuberworm moth Phthorimaea operculella exhibit a series of short and straight intermittent flights, or hops, when moving upwind. It is unclear whether they navigate by employing the same behavioural pattern and wind detection mechanism as that used by flying moths. To analyze odour‐modulated anemotaxis in male potato tuberworm moths, a flat wind tunnel is constructed to give regular odour stimuli to an insect regardless of its location. Moths are subjected to pheromone pulses of different frequencies to test whether they show a behavioural pattern that is reset and restarted by a pheromone pulse. Moths on the ground are also subjected to crosswind shear to examine their detection of wind direction. Path analyses reveal that males surge upwind when they receive a pheromone pulse and exhibit casting by successive hops when they lose odour. This behavioural pattern appears to be similar to that of flying moths. When the direction of the airflow is switched orthogonally, males adjust their course angle accordingly when they are on the ground. It is suggested that, instead of optomotor anemotaxis, this ‘aim‐then‐shoot’ system aids the detection of wind direction, possibly by mechanosensory means.     

Response of muscle-based biochemical condition indices to short-term variations in food availability in post-flexion reared sea bass Dicentrarchus labrax (L.) larvae

Six condition indices based on RNA, total soluble protein and two metabolic enzymes [lactate dehydrogenase (LDH) and citrate synthase (CS)] were analysed in muscle tissue of individual larvae, post-flexion reared sea bass Dicentrarchus labrax using DNA and total soluble protein as standards for size. In addition, the effect of 2 days of food deprivation on the cell proliferation rates was assessed. The RNA:DNA best reflected short-term changes in feeding conditions. If standardized by DNA content, LDH activity was a better indicator of condition than any other index but RNA:DNA. Further, the analysis of cell proliferation rates in muscle from 26 day-old larvae proved useful in distinguishing continuously fed larvae from individuals subjected to 2 days of fast.     

Anti-phospholipase C antibodies inhibit the lectin-induced proliferation of human lymphocytes

A novel approach was used to assess the role of phosphoinositide hydrolysis in the mitogenic action of phytohemagglutinin (PHA) or concanavalin A (ConA). The treatment of human peripheral blood leukocytes (PBL) with monospecific antibodies against phospholipase C (PLC) produced a dose-dependent inhibition (up to 100%) of PHA (10 g/ml) or ConA (25 g/ml) proliferative effects. Thus, the activation of membrane-bound PLC is asine-qua-non condition for lectin-induced proliferation of T lymphocytes. The key-role of PLC versus protein kinase C (PKC) is stressed by the fact that the inhibition of PKC with Hidaka's compound H-7 (40 M) produced only a partial blockade (about 25%) of lectin mitogenic effect.To whom correspondence should be addressed.     

Viral p21 Ki-RAS protein: a potent intracellular mitogen that stimulates adenylate cyclase activity in early G1 phase of cultured rat cells

Rat kidney (NRK) cells infected with a temperature-sensitive mutant of the Kirsten sarcoma virus were arrested in the G0/G1 phase of their cell cycle by incubation in serum-deficient medium at a p21-inactivating temperature of 41 degrees C. These quiescent ts K-NRK cells were then stimulated to transit G1 and initiate DNA replication by lowering the temperature to 36 degrees C, which rapidly reactivated p21. Reactivating the viral Ki-RAS protein by temperature shift led to an increase in adenylate cyclase activity in early G1 phase. The Ki-RAS protein increased the sensitivity of adenylate cyclase to guanyl nucleotides by a mechanism that seemed to involve inactivation of the enzyme's inhibitory G1 regulatory protein.     

In Vivo measurement of unscheduled DNA synthesis and S-phase synthesis as an indicator of hepatocarcinogenesis in rodents

Measurement of chemically induced DNA repair as unscheduled DNA synthesis in rodent liver following in vivo treatment is a useful screen for potential hepatocarcinogens. In addition to measurement of unscheduled DNA synthesis, examination of S-phase synthesis provides an indicator of chemically induced cell proliferation in the liver, which may be a basis for hepatic tumor promotion. Several chemicals and classes of chemicals have been examined using these endpoints. The pyrrolizidine alkaloid riddelline is a potent genotoxic agent in vitro, and in vivo studies confirm this response as riddelline induces significant elevations in unscheduled DNA synthesis and S-phase synthesis in rat liver. Conversely, H. C. Blue dyes #1 and #2 are both potent genotoxic agents in vitro but fail to express this genotoxicity in vivo. H. C Blue #1 induces significant increases in S-phase synthesis in B6C3F1 mouse liver, which correlates with the observed carcinogenicity of this compound. Halogenated hydrocarbons likewise fail to induce unscheduled DNA synthesis in vivo, but many of these compounds do increase hepatic cell proliferation in mice, which may be the principal mechanism of hepatocarcinogenesis in this species.Abbreviations BCMEE bis(2-chloro-l-methylethyl)ether - dThd thymidine - HCB1 H.C. Blue #1 - HCB2 H.C. Blue #2 - UDS unscheduled DNA synthesis     

Development of a liquid flow-through system to inhibit browning in callus cultures of guayule (Parthenium argentatum Gray)

Calli of P. argentatum were grown on a newly designed liquid nutrient flow-through system which facilitated the subculturing of calli and delayed browning for 6 weeks. Friable calli were obtained on half-strength Gamborg B5-medium supplemented with 0.05 mgl⁻¹ 2,4-dichlorophenoxyacetic acid. Shoots developed on media supplemented with 0.2 mgl⁻¹ benzylaminopurine but lacking 2,4-dichlorophenocyacetic acid.