Leukemia 11998) 12, 1724-1 732 © 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 1724 http://www.stockton-press.eo.uk/leu Differentiation and cell-type-restricted expression of HOXC4, HOXCS and HOXC6 m myeloid leukemias and normal myeloid cells 1 2 1 2 1 JJ Bijl , JW van Oostveen , JMM Walboomers', ATP Brink', W Vos , GJ Ossenkoppele and C)LM Meijer ' Department of Pathology and 'Department of Hematology, Academic Hospital of the Vrije Universiteit, Amsterdam, The Netherlands HOX genes have shown a lineage-specific expression in hema­ eage-restricted expression pattern s of HOX genes were found topoiesis and are suggested as being involved in the 5 6 in hematopoietic cells. · HOXC genes are found to be expression of certain adhesion molecules. Recently, we have expressed in human and mouse lymphocytic and myeloid cell demonstrated that HOXC4 and HOXC6, but not HOXC5, are 5 7 9 expressed during lymphoid differentiation. Reports on the lines. • - It appeared that from these genes HOXC8 was expression of these genes in myeloid leukemias and normal ubiquitously expressed in lymphoid, myeloid, erythroid and myeloid cells are still scarce. Therefore, we have investigated megakaryocytic cell lines. Expression of HO XC4 was found the expression of HOXC4, HOXC5 and HOXC6 in purified sub­ to be restricted to lymphoid cells.7 However, the reported data populations of bone marrow in addition to 36 specimens of are obtained by techniques with different sensitivity levels, acute myeloid leukemias (AMLs), eight chronic myeloid leuke­ hindering a reliable comparison. Therefore, we applied a mias (CMLs), several myeloid cell lines and cutaneous localiza­ 10 tions of three myelomonocytic leukemias and one granulocytic sensitive RT-PCR specific for HOXC4, HOXCS and HOXC6. sarcoma by RT-PCR and partly by RNA in situ hybridization Using this specific RT-PCR, we have shown in a previous (RISH). HOXC4 and HOXC6 transcripts were both detected by study that both HOXC4 and HOXC6 are expressed in the RT-PCR in 22/36 and 24/36 AMLs, respectively. The distribution lymphoid lineage.10 HOXC4 mRNA was expressed earlier in of HOXC4 and HOXC6 gene expression over the different types 10 more undifferentiated cells (CD34+/CD38 w cells) th an of AML was largely similar and covered all types of AML In HOXC6 mRNA (prothymocytes and pre-pre B cells), but contrast, HOXC5 gene expression was found in only 6/32 AMLs. Expression of HOXC5 was restricted to AMLs of the granulo­ expression of both genes persisted to maturity. In contrast to cytic (FAB M1-M3), early monocytic (FAB M4) and early HOXC4 and HOXC6, HOXCS expression could not be erythroid (FAB M6) lineage. In general, except in one FAB M5b detected in normal cells of the lymphoid lineage, but only 10 1 1 case, no expression of HOXC5 was found in AMLs derived from in particular types of non-Hodgkin's lymphomas (NHL), , late stages of monocytic (FAB MS) and megakaryocytic (FAB implying a role for HOXCS in tissue positioning of these lym­ M7) lineages. As for HOXC4 and HOXC6, expression of HOXC5 phoma cells. Studies on HOXCS expression in myeloid cell was absent in CMLs. Using RISH significant HOXC4, HOXC5 and HOXC6 expression was found in a number of additionally lines, myeloid leukemias and normal myelo id ce lls are scarce. studied AML samples of different FAB classification (M2, M4, Expression of HOXCS has been found only in some erythroid 5 7 M5b and M5b), (M2 and M5b) (M2, M4, M5b), respectively. In and myeloid cell lines. · tissue localizations of leukemias a different expression pattern These interesting observations prompted us to investigate of HOXC4, HOXC5 and HOXC6 was found. In contrast to mature the expression of HOXCS and that of neighboring genes leukemic stages of myeloid differentiation, these skin localiza­ HOXC4 and HOXC6. For that purpose, we analyzed mono­ tions of leukemias expressed HOXC5 and HOXC6. HOXC4 expression was found both in leukemic cells derived from per­ nuclear cells (M C) derived from bone marrow (BM) aspirates ipheral blood and from cutaneous localizations. Besides obtained from patients with acute and chronic myeloid leuke­ HOXC4 expression in monocytes no expression of HOXC4, mia (AML and CML), myeloid cel l lines and normal granulo­ HOXC5 and HOXC6 was found in granulocytes and monocytes, cytes, monocytes, erythroid colonies of growth factor-induced colonies of growth factor-induced CD34+ bone marrow cells. In CD34+ ce lls and subpopulations of the megakaryocytic lin­ 10 nd earliest CD34·/CD38 w " high cell fractions of bone marrow only eage by RT-PCR and partly by RNA in situ hybrid ization HOXC4 and in megakaryocytic cells both HOXC4 and HOXC6 were found. Thus, the expression patterns of these HOXC (RISH) in order to confirm biologica lly significant expression. genes found in the limited number of cell fractions of normal To characterize the significance of HOXC4, HOXCS and bone marrow suggest that the expression patterns found in HOXC6 expression in tissue localizations we additional ly ana­ AMLs and CMLs might reflect the normal situation. Further­ lyzed cutaneous infiltrates of myelomonocytic leukemias and more, the presence of HOXC5 and HOXC6 expression specifi­ a granulocytic sarcoma. cally in skin infiltrates of late differentiation stages of myeloid leukemias, suggests an additional role for these genes in the positioning of these myeloid cells in skin tissue. Keywords: homeobox genes; expression; myelopoiesis; differen­ Materials and methods tiation; leukemias Isolation of granulocytes, monocytes, erythroid colonies and CFU-MEGA Introduction Blood from normal donors was centrifugated over a Lympho­ Evidence accumulated over the last few yea rs tends to suggest prep density gradient. The fraction containing granulocytes that HOX genes are important geneti c determinants of pro­ was incubated tw ice w ith ammonium-chloride on ice for 10 1 4 liferation an d differen tiation of hematopoietic cells. - Lin- min to lyse the erythrocytes, then washed and pelleted for RNA isolation. Frozen monocytes, isolated by elutriation up to at least 90% purity, were thawed, washed tw ice, counted an d used for RNA isolation. For the generation of erythroid Correspondence: CJLM Meijer, Dept of Pathology, Academic Hospital of the Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, colonies, CD34+ cel ls isolated from normal bone marrow The Netherlands; Fax: 31 20 44 42964 using a miniM acs system (Miltenyi Biotec, Bergisch Gladbach, Received 17 July 1997; accepted 29 April 1998 Germany), were incubated in Methocult GF H4434 (StemCel l Expression of HOXC4, HOXC5 and HOXC6 in myelopoiesis JJ Bijl et al 1725 Technologies, Vancouver, Canada) a methylcellulose (0.9%) Table 1 RT-PCR ana lysis of expression of HOXC4, HOXC5 and containing lscove' s medium supplied with human recombi­ HOXC6 mRNA in bone marrow aspirates from patients with AML •, CML, cell lines and sk in localized myeloid neoplasia nant growth factors (erythropoietin: 3 U/ ml, stem cell factor (SCF) : 50 ng/ml, GM-CSF: 10 ng/ml, and IL-3: 10 ng/ml), at Case HOXC4 HOXC5 HOXC6 U1N 37°C for 2 weeks. Twenty erythroid colonies, recognized by their large red morphology, were picked for RNA isolation. In Undifferentiated cells order to obtain cells of the megakaryocytic lineage, co34+ AML MO 0/2° 0/2 1/2 2/2 cells derived from non-involved BM of a patient with T-NHL 1 + were isolated using miniMacs (Miltenyi Biotec) and cultured 2 + + in lscove's medium supplemented with monothioglycerol, deionised 1 .5% BSA, iron-saturated transferrin (Sigma, St Cells with evidence of granulocytic differentiation Louis, MO, USA), sonicated lipids, 20 ng/ml thrombopoietin AML M1 2/5 1/5 1/5 5/5 3 1 + + + (TPO) (Sigma) and 10 ng/m SCF at 37°C, 5% CO . After 6 2 4-6 + days, cells were harvested and sorted by FACS (Becton 7 + Dickinson, Mountain View, CA, USA) using the membrane AML M2 3/5 1/5 2/5 5/5 marker combinations CD34+/CD41-, CD34+/CD41 + and 8 + + + CD34-/CD41 +. 9 + + + + 10/11 + 12 + + AML M3 3/3 1/3 3/3 3/3 Myeloid leukemias and cell lines 13 + + + + 14 + + + Myeloid cell lines studied were K562 (CML-blast crises), 15 + + + KGla, KGl , THP-1 , promyelocytic cell line HL60 and megak­ CML 0/8 0/8 0/8 8/8 aryocytic cell line M-07e. All cell lines were obtained from 16-23 + American Type Cell Collection (ATCC, Rockville, MD, USA) Cells with evidence of monocytic differentiation and were cultured at 37°C in RPMl-1640 medium with 25 AML M4 3/5 2/5 5/5 5/5 mM Hepes buffer (Gibco BRL, Life Technologies, Gaithers­ 24 + + burg, MD, USA) supplemented with 10% (v/v) heat inacti­ 25 + + vated fetal calf serum (Hyclone Laboratories, Logan, UT, 26 + + + + USA), 1 mM glutamine, 100 U/ml natrium Penicillin G (G ist 27 + + + 28 Brocades NV, Delft, The Netherlands) and 100 U/ml + + + + AML M5a 3/4 0/4 4/4 4/4 Streptomycin Sulphate (Pharmachemie BV, Haarlem, The 29 + + + Netherlands). 30 + + + Snap-frozen or fresh mononuclear cells derived from bone 31 + + marrow aspirates of 36 patients with AML and eight patients 32 + + + with CML were collected from the Department of Hematology AML M5b 1/3 0/3 1/3 3/3 of the Vrije Universiteit Hospital. The AML samples were 33 + + + 34 + ordered according to the criteria adopted from the French­ 35 + American-British (FAB) cooperative group.12 AML samples were routinely characterized for the expression of several Cells with evidence of erythroid differentiation immunomarkers, such as CD34, CD33, CDl 3 and CDl 5 M6 2/4 1/4 3/4 4/4 (Becton Dickinson) by FACS analysis. Cytospins of the cell 36 + + 37 samples were stained with M ay-Grunwald-Giemsa and were + + + + 38 + + + analyzed light-microscopically for the presence of contami­ 39 + nating lymphocytes.