F intercellular tight junctions and typical brush border microvilli projecting perpendicularly to the surface. The expression and activity of brush border enzymes notably alkaline phosphatase (AlkP), are increased in cellular GNF-7 web differentiation [13]. Moreover, the expression of GSTA1 progressively increases asCaco-2 cells differentiate [14]. Terminally differentiated enterocytes undergo apoptosis and are sloughed from the surface epithelium into the intestinal lumen [15]. Therefore apoptosis seems to be a necessary component of colonocyte terminal differentiation. Indeed, neoplastic transformation of the colonic epithelium is associated with disordered regulation of cellular differentiation and apoptosis [16]. Numerous factors are involved in the control of intestinal epithelial cell differentiation, including growth factors, hormones, extracellular matrix proteins, vitamins, and luminal nutrients such as short chain fatty acids [17]. Sodium butyrate (NaB), a shortchain fatty acid present in the human large intestine, is derived from bacterial fermentation of complex carbohydrates. NaB is a preferred energy source for normal colonocytes in vivo but also reduces the growth and motility of colon cancer cell lines and causes dose-dependent cellular differentiation and apoptosis [18,19,20]. GSTs act as mediators of cell signaling kinase pathways involved in cell cycle transition such as proliferation and apoptosis [9]. Progressive increase in GSTA1 expression with cellular confluency in Caco-2 cells may influence responses to cellular stress [14]. Therefore we suspect that GSTA1 may function as a modulator of cell phase transitions. We have previously shown that the incidence of apoptosis stimulated by 15481974 tumour necrosis factor a and sodium butyrate is significantly higher in preconfluent Caco-2 cells with minimal GSTA1 expression compared to differentiatedGSTA1 and Caco-2 Cell Proliferationpostconfluent cells with high GSTA1 expression [14]. We have also demonstrated that GSTA1 forms complexes with c-Jun Nterminal kinase (JNK) and inhibits activation of JNK signalling in Caco-2 cells and that GSTA1 69-25-0 overexpression reduces phosphorylation of c-Jun during oxidative stress [14]. We hypothesized that low GSTA1 expression was a necessary condition for cell proliferation and that increased expression of GSTA1 is a requirement for Caco-2 cell differentiation. Our results indicate that low concentrations (1 mM) of NaB cause Caco-2 cell differentiation and concomitant GSTA1 induction and high concentrations (10 mM) stimulate apoptosis and down-regulation of GSTA1. Moreover, manipulation of GSTA1 levels by forced expression and knockdown using siRNA technology resulted in altered cell proliferation but did not affect NaB-mediated differentiation or sensitivity to apoptosis.5 mM of each primer and 2 mM MgCl2. Oligonucleotide primers for the differentiation makers AlkP were 59 CTCCAACATGGACATTGACG and 39 TGAGATGGGTCACAGACTGG, villin were 59 AGCCAGATCACTGCTGAGGT and 39 TGGACAGGTGTTCCTCCTTC, dippeptidyl peptidase-4 (DDP-4) were 59 GGCGTGTTCAAGTGTGGAAT and 39 TCTTCTGGAGTTGGGAGACC, E-cadherin were 59 TGATCGGTTACCGTGATCAAAA and 39 GTCATCCAACGGGAATGCA and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 59 CAGTCCATGCCATCACTGCC and 39 GCCTGCTTCACCACCTTCTTG. The PCR parameters were 95uC for 1 1662274 min, 1 cycle, and 35 cycles of 95uC for 15 s, 70uC for 5 s and 72uC for 15 s. Messenger RNA levels for these differentiation markers were normalized against GAPDH mRNA.F intercellular tight junctions and typical brush border microvilli projecting perpendicularly to the surface. The expression and activity of brush border enzymes notably alkaline phosphatase (AlkP), are increased in cellular differentiation [13]. Moreover, the expression of GSTA1 progressively increases asCaco-2 cells differentiate [14]. Terminally differentiated enterocytes undergo apoptosis and are sloughed from the surface epithelium into the intestinal lumen [15]. Therefore apoptosis seems to be a necessary component of colonocyte terminal differentiation. Indeed, neoplastic transformation of the colonic epithelium is associated with disordered regulation of cellular differentiation and apoptosis [16]. Numerous factors are involved in the control of intestinal epithelial cell differentiation, including growth factors, hormones, extracellular matrix proteins, vitamins, and luminal nutrients such as short chain fatty acids [17]. Sodium butyrate (NaB), a shortchain fatty acid present in the human large intestine, is derived from bacterial fermentation of complex carbohydrates. NaB is a preferred energy source for normal colonocytes in vivo but also reduces the growth and motility of colon cancer cell lines and causes dose-dependent cellular differentiation and apoptosis [18,19,20]. GSTs act as mediators of cell signaling kinase pathways involved in cell cycle transition such as proliferation and apoptosis [9]. Progressive increase in GSTA1 expression with cellular confluency in Caco-2 cells may influence responses to cellular stress [14]. Therefore we suspect that GSTA1 may function as a modulator of cell phase transitions. We have previously shown that the incidence of apoptosis stimulated by 15481974 tumour necrosis factor a and sodium butyrate is significantly higher in preconfluent Caco-2 cells with minimal GSTA1 expression compared to differentiatedGSTA1 and Caco-2 Cell Proliferationpostconfluent cells with high GSTA1 expression [14]. We have also demonstrated that GSTA1 forms complexes with c-Jun Nterminal kinase (JNK) and inhibits activation of JNK signalling in Caco-2 cells and that GSTA1 overexpression reduces phosphorylation of c-Jun during oxidative stress [14]. We hypothesized that low GSTA1 expression was a necessary condition for cell proliferation and that increased expression of GSTA1 is a requirement for Caco-2 cell differentiation. Our results indicate that low concentrations (1 mM) of NaB cause Caco-2 cell differentiation and concomitant GSTA1 induction and high concentrations (10 mM) stimulate apoptosis and down-regulation of GSTA1. Moreover, manipulation of GSTA1 levels by forced expression and knockdown using siRNA technology resulted in altered cell proliferation but did not affect NaB-mediated differentiation or sensitivity to apoptosis.5 mM of each primer and 2 mM MgCl2. Oligonucleotide primers for the differentiation makers AlkP were 59 CTCCAACATGGACATTGACG and 39 TGAGATGGGTCACAGACTGG, villin were 59 AGCCAGATCACTGCTGAGGT and 39 TGGACAGGTGTTCCTCCTTC, dippeptidyl peptidase-4 (DDP-4) were 59 GGCGTGTTCAAGTGTGGAAT and 39 TCTTCTGGAGTTGGGAGACC, E-cadherin were 59 TGATCGGTTACCGTGATCAAAA and 39 GTCATCCAACGGGAATGCA and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 59 CAGTCCATGCCATCACTGCC and 39 GCCTGCTTCACCACCTTCTTG. The PCR parameters were 95uC for 1 1662274 min, 1 cycle, and 35 cycles of 95uC for 15 s, 70uC for 5 s and 72uC for 15 s. Messenger RNA levels for these differentiation markers were normalized against GAPDH mRNA.
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