John C. Livesey

Differential regulation of calcium homeostasis in adenocarcinoma cell line A549 and its Taxol‐resistant subclone

Drug resistance is a fundamental problem in cancer chemotherapy. Intracellular calcium concentration ([Ca2+]i) may play a role in the development of chemoresistance. We investigated the regulatory role of [Ca2+]i in Taxol resistance in the non‐small‐cell lung cancer cell line A549 and its chemoresistant subclone A549‐T24. Measurement of cytosolic calcium ([Ca2+]c) in single cells and cell populations revealed similar levels of basal calcium in the two cell lines. However, a reduced response to thapsigargin (a sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor) in A549‐T24 cells compared to the parent cell line suggested a lower ER Ca2+ content in these cells. mRNA expression of SERCA2b and SERCA3, major Ca2+ pumps involved in ER Ca2+ homeostasis, did not significantly differ between the two cell lines, as revealed by RT–PCR. An altered calcium influx pathway in the Taxol‐resistant cell line was observed. Modulation of the ER calcium pools using CMC (4‐chloro‐m‐cresol) and ATP revealed lower ryanodine receptor (RyR) and IP3 receptor (IP3R)‐sensitive Ca2+ stores in the chemoresistant cell line. Western blot and RT–PCR studies suggested that A549‐T24 cells expressed higher levels of the antiapoptotic protein Bcl‐2 and the calcium‐binding protein sorcin, respectively, in comparison to the parent cell line. Both of these proteins have been previously implicated in chemoresistance, in part, due to their ability to modulate [Ca2+]i. These results suggest that altered intracellular calcium homeostasis may contribute to the Taxol‐resistant phenotype.

AKT-Induced Tamoxifen Resistance Is Overturned by RRM2 Inhibition

Acquired tamoxifen resistance develops in the majority of hormone-responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biologic outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNA interference (RNAi)–mediated approaches significantly reversed the tamoxifen-resistant cell growth, inhibited cell motility, and activated DNA damage and proapoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM inhibitor didox significantly reduced in vitro and in vivo growth. Thus, AKT-expressing breast cancer cells upregulate RRM2 expression, leading to increased DNA repair and protection from tamoxifen-induced apoptosis. Implications: These findings identify RRM2 as an AKT-regulated gene, which plays a role in tamoxifen resistance and may prove to be a novel target for effective diagnostic and preventative strategies. Mol Cancer Res; 12(3); 394–407. ©2013 AACR.

An evaluation of a human stem cell line to identify risk of developmental neurotoxicity with antiepileptic drugs

Determination of the impact of a drug on human brain development relies instead on surrogate animal studies. Here we have exploited the human stem cell line, TERA2.cl.SP12 to differentiate into neurons and addressed their value as an in vitro model to evaluate the risk of developmental neurotoxicity with antiepileptic drugs (AEDs). The effects of four AEDs were investigated on cell viability, cell cycle and neural differentiation. Exposure to either phenobarbital (10-1000 μM), valproic acid (10-1000 μM), lamotrigine (1-100 μM) or carbamazepine (1-100 μM) for 3 days reduced viability in non-differentiating cells only at the highest concentrations tested. Viability was also reduced with lower concentrations of all AEDs in cells undergoing neural differentiation. Valproic acid and carbamazepine increased DNA fragmentation and reduced cell cycle progression. 3 days exposure at the start of neural differentiation to phenobarbital, valproic acid or lamotrigine also significantly reduced the proportion of stem cells that subsequently differentiated into neurons at 15 days in vitro. The two control agents tested, ciprofloxacin and perfluorooctanoic acid had no impact on neurogenesis in vitro. These new data show that modelling neurogenesis in vitro using a human stem cell line may be a powerful method to predict risks of developmental neurotoxicity in vivo with psychotropic drugs.