Transcriptomes reflect the phenotypes of undifferentiated, granulocyte and macrophage forms of HL-60/S4 cells

Abstract

To understand the chromatin changes underlying differential gene expression during induced differentiation of human leukemic HL-60/S4 cells, we conducted RNA-Seq analysis on quadruplicate cultures of undifferentiated, granulocytic- and macrophage-differentiated cell forms. More than half of mapped genes exhibited altered transcript levels in the differentiated cell forms. In general, more genes showed increased mRNA levels in the granulocytic form and in the macrophage form, than showed decreased levels. The majority of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in genes that exhibited differential transcript levels after either RA or TPA treatment. Changes in transcript levels for groups of genes with characteristic protein phenotypes, such as genes encoding cytoplasmic granular proteins, nuclear envelope and cytoskeletal proteins, cell adhesion proteins, and proteins involved in the cell cycle and apoptosis illustrate the profound differences among the various cell states. In addition to the transcriptome analyses, companion karyotyping by M-FISH of undifferentiated HL-60/S4 cells revealed a plethora of chromosome alterations, compared with normal human cells. The present mRNA profiling provides important information related to nuclear shape changes (e.g., granulocyte lobulation), deformability of the nuclear envelope and linkage between the nuclear envelope and cytoskeleton during induced myeloid chromatin differentiation.

Keywords: Acute myeloid leukema; apoptosis; cell attachment; cell differentiation; cell division; cytoskeleton; granulocyte; karyotype; mRNA levels; macrophage; nuclear envelope.

Figure 1.

Differential transcript levels for cytoplasmic granular proteins. Bars indicate the ratio of RSEM (RNA-Seq by Expectation-Maximization) expected counts of treatment (RA or TPA) compared with control (undifferentiated HL-60/S4 cells). Gene names are HUGO Gene Nomenclature Committee (HGNC) gene symbols; repetition of the gene name indicates multiple isoforms of the gene. Bars indicate difference in transcript levels from untreated control: solid bars indicate differential transcript levels with posterior probability ≥ 0.95; open bars indicate posterior probability < 0.95. Asterisks indicate that the transcript level after one treatment is significantly different than the other treatment (p.p. ≥ 0.95), when both exhibit increased or decreased trancript levels. Only isoforms where the total RSEM expected count was ≥ 10 across all 3 cell states, or where the contribution of the isoform to the total count of the gene was ≥ 0.05 for at least one condition, are shown. Details for all isoforms, including NCBI refseq identifiers, are in Supplemental Table 1.

Figure 2.

Differential transcript levels for nuclear envelope and cytoskeletal proteins. Graphical representation is the same as described in the legend for Fig. 1. Details for all isoforms, including NCBI refseq identifiers, are in Supplemental Table 2.

Figure 3.

Differential transcript levels for proteins involved in the cell cycle. (A) Cyclins; (B) Cyclin Dependent Kinases; (C) CDK Inhibitors. Graphical representation is the same as described in the legend for Fig. 1. Details for all isoforms, including NCBI refseq identifiers, are in Supplemental Table 3.

Figure 4.

Differential transcript levels for proteins involved in apoptosis. (A) top, initiator caspases; bottom, caspase 1 and effector caspases. (B) Other genes involved in Apoptosis. Graphical representation is the same as described in the legend for Fig. 1. Details for all isoforms, including NCBI refseq identifiers, are in Supplemental Table 4.

Figure 5.

Light microscopic images of HL-60/S4 cells following treatment with 16 nM TPA. Cell attachment and progressive cell clustering are readily apparent. “Day,” refers to the days after addition of TPA. Magnification bar, 100 µm.

Figure 6.

Differential transcript levels for proteins involved in cell attachment. KEGG Pathway: “Extracellular Matrix (ECM) Receptor Interactions.” Graphical representation is the same as described in the legend for Fig. 1. Details for all isoforms, including NCBI refseq identifiers, are in Supplemental Table 5.

Figure 7.

Differential transcript levels for proteins involved in cell attachment. KEGG Pathways: “Adherens Junctions” and “Cell Adhesion Molecules.” Graphical representation is the same as described in the legend for Fig. 1. Details for all isoforms, including NCBI refseq identifiers, are in Supplemental Table 5.

Figure 8.

Differential transcript levels for proteins involved in cell attachment. KEGG Pathway: “Leukocyte Transendothelial Migration.” Graphical representation is the same as described in the legend for Fig. 1. Details for all isoforms, including NCBI refseq identifiers, are in Supplemental Table 5.

Figure 9.

M-FISH Karyotype Analysis of HL-60/S4 Cells. Top, representative multicolor karyogram of undifferentiated HL-60/S4 cells. Each chromosome is presented in a specific classification color allowing the easy detection of numerical and structural chromosome aberrations. Bottom, inverted DAPI staining results in a GTG-like banding pattern of the chromosomes. Data in Supplemental Table 8.