Expressed sequence tag (EST) phenotyping of HT-29 cells: cloning of ser/thr protein kinase EMK1, kinesin KIF3B, and of transcripts that include Alu repeated elements

To study the mechanisms that control epithelial commitment and differentiat ion we have used undifferentiated HT-29 colon cancer cells and a subpopulat ion of mucus secreting cells obtained by selection of HT-29 cells in 10(-6) M methotrexate (M6 cells) as experimental models. We isolated cDNAs encodi ng transcripts overexpressed in early confluent M6 cells regarding steady-s tate levels in HT-29 cells by subtractive hybridisation. Fifty-one cDNA clo nes, corresponding to 34 independent transcripts, were isolated, partially sequenced by their 5' end, and classified into four groups according to the ir identity: transcripts that included a repeated sequence of the Alu famil y (10 clones, among them those encoding ribonucleoprotein RNP-L and E-cadhe rin), transcripts encoded by the mitochondrial genome (nine clones), transc ripts encoding components of the protein synthesis machinery (23 clones, in cluding the human ribosomal protein L38 not previously cloned in humans) an d nine additional cDNAs that could not be classified in the previous groups . These last included ferritin, cytokeratin 18, translationally controlled human tumour protein (TCHTP), mt-aldehyde dehydrogenase, as well as unknown transcripts (three clones), and the human homologues of the molecular moto r kinesin KIF3B and of the ser/thr protein kinase EMK1. Spot dot and Northe rn blot analyses showed that ser/thr protein kinase EMK was differentially expressed in M6 cells when compared with parental HT-29 cells. Steady-state levels of EMK1 were higher in proliferating, preconfluent, M6 and HT-29 ce lls than in 2 days post confluence (dpc) and 8dpc M6 and HT-29 cells. Trans cripts that included an Alu repeat were also shown to be differentially exp ressed and accumulated in differentiating M6 cells when analysed by Norther n blot. The significance of the transcripts cloned is discussed in the cont ext of the commitment and differentiation of the M6 cells to the mucus secr eting lineage of epithelial cells. (C) 1999 Elsevier Science B.V. All right s reserved.