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All trans-retinoic acid (ATRA) can induce granulocytic differentiation both in vitro and in vivo, and its activity is mediated by the retinoic acid receptor-alpha (RAR-alpha). In the present study, we evaluated the ability of this inducer in HL-60 cells, to stimulate simultaneously granulocytic differentiation and the expression of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-3, IL-6, tumor necrosis factor-alpha (TNF-alpha), and stem cell factor (SCF). The level of expression of these cytokines in ATRA-treated HL-60 cells was compared with that observed in normal and lipopolysaccharide (LPS)-treated peripheral granulocytes. The results indicate that the expression of these cytokines is enhanced during differentiation so that the pattern observed in ATRA-treated HL-60 cells is close to that of LPS-stimulated normal granulocytes. In addition, tetra phorbol acetate (TPA)-treated HL-60 cells express several of the above listed cytokines. It is concluded that ATRA not only induces granulocytic differentiation of HL-60 cells, but also activation of these terminally differentiated cells. The activating cytokine expression in these cells appears related to the progress of the differentiation program induced by ATRA since normal granulocytes do not respond to this inducer by activation of the expression of these genes. Furthermore, the cytokine activation is a specific effect of ATRA, since DMSO does not have any stimulatory effect.

The relative abundance of primary transcript and mature mRNA of the c-myc, calcyclin, S14 ribosomal protein, and rRNA genes was determined densitometrically after reverse transcriptase-polymerase chain reaction and Northern blotting analysis in resting and mitogen-stimulated lymphocytes, proliferating and terminally differentiated HL-60 cells, and leukemic blast cells. Transcription and processing of c-myc and rRNA gene transcripts increased proportionally after mitogen stimulation, whereas these processes were independent of cell cycling status in the case of the S14 gene. Normal lymphocytes showed an unexpectedly large amount of primary transcript of the calcyclin gene, whereas the corresponding mRNA was undetectable. The abundance of c-myc, calcyclin, and S14 mRNA in terminally differentiated HL-60 cells decreased sharply, compared to their proliferating counterparts. This decrease reflected post-transcriptional modulation, since the abundance of precursor remained essentially unchanged. After HL-60 differentiation, the 32S rRNA levels remained relatively high, whereas the 45S primary transcript almost disappeared. Leukemic blast cells displayed highly variable precursor/mRNA ratios of c-myc, calcyclin, and S14 genes but consistently high ratios of 32S to 45S RNA, suggesting that the cleavage rate of the 32S rRNA was sharply reduced in these cells, resulting in an accumulation of this molecule. These results suggest the importance of efficient processing of primary transcript to generate adequate functional mRNA, thus regulating gene expression. Furthermore, in terminally differentiated cells and leukemic blast cells a stabilization of the primary transcript without efficient processing can be observed. The role of the stabilization of the primary transcript in terminal differentiation is further supported by the results of run-off transcription, indicating a sharp decrease of c-myc and calcyclin transcription rate in retinoic acid/dimethyl sulfoxide-treated HL-60 cells.

The hybridization to a complementary RNA (cRNA) probe both in situ and in solution was used to assay tiny amounts of mRNA of the lactoferrin (LF) and myeloperoxidase (MPO) genes in normal bone marrow cells and in acute and chronic lymphoid leukemias. Evidence is reported that this technique is much more sensitive than the standard Northern blot technique. The LF mRNA was detectable in three of seven cases of acute lymphoblastic leukemia (ALL) and in three of seven cases of chronic lymphocytic leukemia (CLL). Four cases of ALL were also positive when tested with the MPO cRNA. It is apparent from these results that myeloid specific mRNA, different from MPO, may be detected in leukemic cells with lymphoid phenotype using a method more sensitive than the Northern blot technique. Whether or not the molecular events observed in these cell populations reflect events physiologically occurring rather than a deregulation of gene expression associated to leukemogenesis remains to be established.

Whole-cell RNA, extracted from acute myeloid leukemia blast cells, was fractionated by sedimentation through sucrose gradients. The proportion of double-helical segments present in each fraction was then determined by a quantitative microcomplement fixation assay that specifically measures double-helical RNA. Sizable amounts of double-helical segments were detected in all fractions of cellular RNA corresponding to S values higher than approximately 20. In all cell populations examined the highest proportion of double-helical segments was found in RNA fractions sedimenting faster then the 45 S ribosomal precursors RNA, i.e., in fractions including only heterogeneous nuclear RNA.