Murine Cell Line 32D Research Articles

Differential roles of STAT1 and STAT2 in the sensitivity of JAK2V617F- vs. BCR-ABL-positive cells to interferon alpha

BackgroundInterferon alpha (IFNa) monotherapy is recommended as the standard therapy in polycythemia vera (PV) but not in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa efficacy in JAK2V617F- vs. BCR-ABL-positive cells.MethodsGene expression microarrays and RT-qPCR of PV vs. CML patient PBMCs and CD34+ cells and of the murine cell line 32D expressing JAK2V617F or BCR-ABL were used to analyze and compare interferon-stimulated gene (ISG) expression. Furthermore, using CRISPR/Cas9n technology, targeted disruption of STAT1 or STAT2, respectively, was performed in 32D-BCR-ABL and 32D-JAK2V617F cells to evaluate the role of these transcription factors for IFNa efficacy. The knockout cell lines were reconstituted with STAT1, STAT2, STAT1Y701F, or STAT2Y689F to analyze the importance of wild-type and phosphomutant STATs for the IFNa response. ChIP-seq and ChIP were performed to correlate histone marks with ISG expression.ResultsMicroarray analysis and RT-qPCR revealed significant upregulation of ISGs in 32D-JAK2V617F but downregulation in 32D-BCR-ABL cells, and these effects were reversed by tyrosine kinase inhibitor (TKI) treatment. Similar expression patterns were confirmed in human cell lines, primary PV and CML patient PBMCs and CD34+ cells, demonstrating that these effects are operational in patients. IFNa treatment increased Stat1, Stat2, and Irf9 mRNA as well as pY-STAT1 in all cell lines; however, viability was specifically decreased in 32D-JAK2V617F. STAT1 or STAT2 knockout and reconstitution with wild-type or phospho-deficient STAT mutants demonstrated the necessity of STAT2 for IFNa-induced STAT1 phosphorylation in BCR-ABL- but not in JAK2V617F-expressing cells. STAT1 was essential for IFNa activity in both BCR-ABL- and JAK2V617F-positive cells. Furthermore, ChIP experiments demonstrate higher repressive and lower active chromatin marks at the promoters of ISGs in BCR-ABL-expressing cells.ConclusionsJAK2V617F but not BCR-ABL sensitizes MPN cells to interferon, and this effect was dependent on STAT1. Moreover, STAT2 is a survival factor in BCR-ABL- and JAK2V617F-positive cells but an IFNa-sensitizing factor solely in 32D-JAK2V617F cells by upregulation of STAT1 expression.

5-Azacytidine reactivates the erythroid differentiation potential of the myeloid-restricted murine cell line 32D Ro

32D cells grown for 1 year in interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) generated the 32D Ro cell line which retained the parental mast cell phenotype but lost ability to generate erythroid cells in response to erythropoietin (EPO). In order to clarify the mechanisms underlying such restriction, we compared 32D and 32D Ro cells for their capacity to express erythroid-specific transcription factors ( Gata1, Gata2, Scl, Nef2, Eklf, and Id) and the capacity of short exposure to 5-azacytidine (5-AzaC) to reactivate erythroid differentiation potential in 32D Ro cells. By Northern analysis, the two cell lines expressed similar levels of all these genes. However, after being treated with 5-AzaC, 32D Ro cells acquired the ability to generate EPO-dependent clones (1 clone/10 4 cells) which gave rise to EPO-dependent cell lines. All the 10 EPO-responsive cell lines independently isolated from 5-AzaC-treated 32D Ro cells had erythroid morphology and expressed high levels of α- and β-globin. In contrast, none of the IL-3-dependent and granulocyte/macrophage colony-stimulating factor-dependent clones concurrently isolated, as a control, showed erythroid properties. Therefore, 5-AzaC treatment reactivates the potential of the myeloid-restricted 32D Ro cells to generate EPO-responsive erythroid clones suggesting that gene methylation played an important role in the G-CSF-mediated restriction/activation of the differentiation potential of these cells.

Somatostatin modulates G-CSF-induced but not interleukin-3-induced proliferative responses in myeloid 32D cells via activation of somatostatin receptor subtype 2

Somatostatin, originally identified as a peptide involved in neurotransmission, functions as an inhibitor of multiple cellular responses, including hormonal secretion and proliferation. Somatostatin acts through activation of G-protein-coupled receptors of which five subtypes have been identified. We have recently established that human CD34/c-kit expressing hematopoietic progenitors and acute myeloid leukemia (AML) cells exclusively express SSTR2. A major mechanism implicated in the antiproliferative action of somatostatin involves activation of the SH2 domain-containing protein tyrosine phosphatase SHP-1. While 0.1-1 x 10(-9) M of somatostatin, or its synthetic stable analog octreotide, can inhibit G-CSF-induced proliferation of AML cells, little or no effects are seen on GM-CSF- or IL-3-induced responses. To study the mechanisms underlying the antiproliferative responses of myeloblasts to somatostatin, clones of the IL-3-dependent murine cell line 32D that stably express SSTR2 and G-CSF receptors were generated. Similar to AML cells, octreotide inhibited G-CSF-induced but not IL-3-induced proliferative responses of 32D[G-CSF-R/SSTR2] cells. Somatostatin induced SHP-1 activity and inhibited G-CSF-induced, but not IL-3-induced, activation of the signal transducer and activator of transcription proteins STAT3 and STAT5. Based on these data and previous results, we propose a model in which recruitment and activation of the tyrosine phosphatase SHP-1 by SSTR2 is involved in the selective negative action of somatostatin on G-CSF-R signaling.

Transcriptional Regulation of Two Myeloid-Specific Genes, Myeloperoxidase and Lactoferrin, During Differentiation of the Murine Cell Line 32D C13

The transcriptional regulation of myeloperoxidase (MPO) and lactoferrin (LF) was examined during terminal myeloid differentiation of the murine cell line 32D C13. The rates of transcription initiation for MPO and LF, determined by an in vitro nuclear run-on assay, increased approximately ninefold. The accumulation of MPO mRNA in 32D C13 cells, determined by Northern blot analysis, correlated temporally with the observed increase in MPO transcription initiation. On the other hand, accumulation of LF mRNA lagged behind the observed increase in LF transcription initiation. In mouse L cells, the LF gene was transcribed more frequently than the MPO gene, though neither mRNA accumulated. Finally, murine MPO transcription is shown, by Northern blot and primer extension analysis, to initiate at multiple sites. These results indicate that whereas transcription induction may largely account for the accumulation of MPO mRNA during terminal myeloid differentiation, both transcriptional and posttranscriptional mechanisms operate to allow accumulation of LF mRNA. The 32D C13 cell system will be a useful model for elucidating these mechanisms.

Transcriptional regulation of two myeloid-specific genes, myeloperoxidase and lactoferrin, during differentiation of the murine cell line 32D C13

The transcriptional regulation of myeloperoxidase (MPO) and lactoferrin (LF) was examined during terminal myeloid differentiation of the murine cell line 32D C13. The rates of transcription initiation for MPO and LF, determined by an in vitro nuclear run-on assay, increased approximately ninefold. The accumulation of MPO mRNA in 32D C13 cells, determined by Northern blot analysis, correlated temporally with the observed increase in MPO transcription initiation. On the other hand, accumulation of LF mRNA lagged behind the observed increase in LF transcription initiation. In mouse L cells, the LF gene was transcribed more frequently than the MPO gene, though neither mRNA accumulated. Finally, murine MPO transcription is shown, by Northern blot and primer extension analysis, to initiate at multiple sites. These results indicate that whereas transcription induction may largely account for the accumulation of MPO mRNA during terminal myeloid differentiation, both transcriptional and posttranscriptional mechanisms operate to allow accumulation of LF mRNA. The 32D C13 cell system will be a useful model for elucidating these mechanisms.

Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D.

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.