The role of pH on differential gene expression to define functionality

INTRODUCTION: Adaptation to environmental changes is a foundational characteristic of all life. Along with temperature, oxygen concentration and many other variables, relative pH is a key factor that cells must regulate in order to maintain viability and functionality. Studies have shown upregulated gene expression of acid tolerance response genes in bacteria when exposed to high acidity. Research has also linked significant pH changes in extracellular environment to different diseases and the cells subsequent responses. Acidosis is a hallmark of both in tumors of various cancers and in inflammation. Previous studies have found solid tumors maintain acidic extracellular pH due to increased respiration associated with glycolysis due to lack of proper vascularization which decreases oxygen saturation in the tumor. Other studies have demonstrated that acidic pH promotes expression of pro-apoptotic and pro-inflammatory cytokines. Many studies have also focused on pH as it pertains to specific pathologies associated with disease conditions. Despite the limited progress, research on the effects of the acidic environment on broader gene expression patterns corresponding to critical signaling pathways that are targeted for various treatment strategies remain unexplored. OBJECTIVE: To evaluate the effect of microenvironmental acidity on regulation of gene expression related to regulating transcription in epithelials, human embryonic kidney cells and human fibroblasts. METHODS: Human embryonic kidney (HEK293), immortal breast MCF10A, fibroblast (HDFa) and cancerous (HCT116) cells were grown in stock medium. Cells were cultured in 1mL wells under control, 12.5 µL of 0.2M HCl/mL, 25 µL of 0.2M HCl/mL and 37.5 µL of 0.2M HCl/mL of culture. Cells were collected after 3 days under experimental conditions. RNA and DNA were collected and purified usingDirect-Zol DNA/RNA Miniprep Kit. For RT-qPCR analysis, we synthesized cDNA from eluted RNA via SuperScript™ IV First-Strand Synthesis System, while concentrations of DNA, RNA and cDNA of each sample was determined via Nanodrop U-Vis spectrophotometer. Expression levels of neurotrophic tyrosine receptor kinase 2 (NTRK2), DNA (cytosine-5)-methyltransferase 1 & 3α (DNMT1, 3A) and Transforming Growth Factor Beta 2 & 1 (TGFB2, 1) were determined by RT-PCR analysis using Bio Rad CFX-Manager Program. The expression levels of sirtuin-silent-mating type information regulation 2 homolog-1 (SIRT1) was also tested for but only in the cancer cell line. Note that the selection of this group of genes was based on our recent studies that provided evidence for the expression of genes such as TGFB2, NTRK2 and DNMT3A exhibiting inverse correlation to pH in an expression microarray analysis of human post-mortem brain samples. RESULTS: Decreasing environmental pH did not directly indicate increased cellular replication as DNA and RNA concentrations in collected samples had no significant changes as extracellular pH increased. Strong association between decreased extracellular pH and increased gene expression was not unilateral across all tested genes and cell types. Increased DMNT1 expression directly correlated w/ decreased pH in all tested cell types. DMNT3A and TGFB1 increased in HDFa and HEK293; TGFB2 expression increased while TGFB1 expression exhibited no change as pH decreased in MCF10A cells. SIRT1 expression remained unchanged under any acid condition in the HCT116 cell line. Our data showed a strong correlation between decreased pH and increased NTRK2 expression in HEK293 and HDFa cells. Furthermore, we found no evidence for altered expression of these genes as well as SIRT1 in the cancer cell line. DISCUSSION: The increased expression of cytokines such as TGFB1/2 and DNMTs affecting transcription indicates that non-cancerous cells have an overall increased gene expression in response to acidic pH. Research procedures should be revisited for optimization using more normal and cancer cell lines to eliminate factors potentially responsible for the inconsistencies in altered gene expression patterns in order to establish their importance in disease pathogenesis and for exploring novel therapeutic remedies