Calcitonin receptor gene expression in K562 chronic myelogenous leukemic cells

Abstract

BACKGROUND: The peptide hormone calcitonin (CT) can significantly effect the proliferation rate of CT receptor (CTR) positive human cancer cells. We wish to identify additional human cancers expressing CTRs and assay the effects of CT on their growth rates and signal transduction pathways. RESULTS: The expression of the human calcitonin receptor (hCTR) gene in the chronic myelogenous leukemia cell line K562 was examined. RT-PCR on total RNA extracted from K562 cells detected the presence of hCTR mRNA. Further analysis demonstrated that multiple hCTR isoforms were present. Incubation of K562 cells with salmon calcitonin (sCT), but not amylin, caused an increase in intracellular levels of cAMP similar to that induced by forskolin treatment. We further demonstrated that butyrate induced erythroid differentiation of K562 cells caused a significant decrease in hCTR mRNA levels. However, phorbol myristate acetate (PMA) induced megakaryocytic differentiation of these cells had no significant effect on hCTR mRNA levels. We demonstrated that exposure to various concentrations of sCT had no effect on the cellular proliferation of K562 cells in vitro. CONCLUSION: Chronic myelogenous k562 cells express multiple CTR isoforms. However, CT does not effect K562 proliferation rates. It is likely that the small increase in intracellular levels of cAMP following CT treatment is not sufficient to interfere with cellular growth.

Figure 1

Southern blot analysis of RT-PCR products. RT-PCR and no RT controls were carried out on total RNA from K562 cells, T47D (hCTR positive) and 3T3 (hCTR negative) cells employing hCTR and actin specific oligos. PCR products were size fractionated on a 1 % agarose gel and Southern blotted. hCTR and actin specific ³²P end labelled probes were hybridized to blot. These results are representative of 3 independent experiments all giving similar results.

Figure 2

Southern blot analysis on gel purified K562 RT-PCR products. (A) RT-PCR products on total RNA from K562 cells employing hCTR specific primers were size fractionated on a 2% agarose gel. Two bands, corresponding in size to hCTR_1a and hCTR_1b, were individually purified and re-run on a 2% agarose gel followed by Southern blot analysis. The blot was hybridized with a ³²P end-labelled probe common to hCTR_1a and hCTR_1b. (B) Blot in Fig. 2A was stripped of probe and re-hybridized with a ³²P end-labelled probe corresponding to 16 amino acid insert in hCTR_1b isoform. These results are representative of 2 independent experiments giving similar results.

Figure 3

Semi-quantitative RT-PCR analysis on butyrate and PMA treated K562 cells. Total RNA from butyrate, PMA and untreated K562 cells was reverse transcribed and subjected to 30, 35 and 40 cycles of PCR employing actin and hCTR specific oligos. Reaction products were size fractionated on an agarose gel and Southern blotted employing ³²P end labelled hCTR and actin specific probes. These results are representative of 3 independent experiments giving similar results.

Figure 4

Intracellular levels of cAMP in amylin, sCT and forskolin treated K562 cells. K562 cells were treated with varying concentrations of amylin and sCT for 20 minutes followed by analysis of intracellular cAMP levels. Forskolin, a known activator of cAMP, was employed as a positive control. Each data point represents an average of 2 assays. These results are representative of 2 independent experiments each giving similar results.

Figure 5

Cellular proliferation of sCT treated K562 cells. K562 cells were treated with varying concentrations of sCT and assayed daily over a four-day period for cell number. Each data point represents an average of four cell counts. These results are representative of 3 independent experiments each giving similar results.