Cell Biochem Biophys (2012) 63:133–141 DOI 10.1007/s12013-012-9349-y

ORIGINAL PAPER

Studies on HOXB4 Expression During Differentiation of Human Cytomegalovirus-infected Hematopoietic Stem Cells into Lymphocyte and Erythrocyte Progenitor Cells Liu Wen-jun • Guo qu-lian • Chen Hong-ying Zou Yan • Huang Mei-xian

•

Published online: 9 March 2012 Ó Springer Science+Business Media, LLC 2012

Abstract We investigated the role of homeobox B4 (HOXB4) mRNA/protein expression induced by human cytomegalovirus (HCMV) and/or all-trans retinoic acid (ATRA) in proliferation and committed differentiation of human cord blood hematopoietic stem cells (HSCs) into colony-forming-units of T-lymphocyte (CFU-TL) and erythroid (CFU-E) progenitors in vitro. Twelve cord blood samples were collected from the fetal placenta umbilical vein and cultured in vitro. The proliferation and differentiation of cord blood HSCs into CFU-TL and CFU-E were continuously disrupted with HCMV-AD169 and/or 6 9 10-8 mol/l of ATRA. HOXB4 mRNA/protein expression in CFU-TL and CFU-E was detected in control, ATRA, HCMV and ATRA ? HCMV groups on days 3, 7, and 12 of culture by fluorescent qRT-PCR/western blot. We found that HOXB4 mRNA/protein expression was detectable on day 3, increased on day 7 and was highest on day 12. HOXB4 mRNA/protein expression in HCMV group was downregulated compared with control group (P \ 0.05). However, the levels were significantly upregulated in HCMV ? ATRA group compared with HCMV group (P \ 0.05). We concluded that the abnormal HOXB4 mRNA/protein expression induced by HCMV could play a role in hematopoietic damage. ATRA, at the concentration used, significantly up-regulated HOXB4 mRNA/protein expression in normal lymphocyte and erythrocyte progenitor cells as well as in HCMV-infected cells. Keywords HOXB4  Cytomegalovirus  Lymphocyte progenitor cells  Erythrocyte progenitor cells

L. Wen-jun (&)  G. qu-lian  C. Hong-ying  Z. Yan  H. Mei-xian Department of Pediatrics, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, Sichuan Province, China e-mail: [email protected]

Introduction Human cytomegalovirus (HCMV) infection can cause severe hematopoietic functional lesions, such as mononucleosis, lymphocytosis, splenohepatomegalia, thrombocytopenic purpura, hemolytic anemia, and bone marrow depression. HCMV, together with toxoplasma, rubella virus, herpes simplex virus, and Treponema pallidum are regarded as five biological teratogenic factors. Numerous studies have shown that the hematopoietic system is one of the major organs suffering from HCMV infection. The hematopoietic stem cells (HSCs) are the major target cells and the major recipient cells for HCMV infection. The pathophysiology of HCMV latency and the nature of HCMV interaction with hematopoietic cells remain unknown. It was reported that HCMV directly damaged the hematopoietic progenitor cells and led to abnormal homeobox (Hox) gene expression in infected cells [1–4]. Hox gene is associated with the development of hematopoietic cells and directly affects the proliferation, differentiation and maturity of hematopoietic stem cells due to structural and functional alterations. HOXB4 is considered to be the major regulatory factor in the proliferation and differentiation of hematopoietic progenitor cells [5, 6]. Heise et al. [7] reported that HCMV infection often fractured some chromosome sites, and one of the common sites was 17q21–22. Interestingly, the HOX gene B cluster is located at chromosome 17. Hox genes expression can be regulated by all-trans retinoic acid (ATRA). However, it is not clear: (i) how HCMV inhibits hematopoiesis; (ii) how HCMV/ATRA regulates HOXB expression; and (iii) how the damage to the hematopoietic system is caused. In this study, we evaluated the role of HXB4 mRNA and protein expression in the proliferation and committed differentiation of human cord blood HSCs into colony forming unit-T-lymphocyte

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(CFU-TL) and colony forming unit-erythroid (CFU-E) precursors as well as the mechanism of damage to cord blood HSCs by HCMV infection.

Materials and methods Samples Placental cord blood samples from 12 full-term infants were collected at the Department of Obstetrics and Gynecology, Affiliated Hospital of Luzhou Medical College, Sichuan, China. All new mothers were in good health and were HBsAg negative. The indices of serum anti-HCMVIgG and -HCMV-IgM of samples detected by ELISA and HCMV-DNA assessed by PCR were negative. HCMVAD169 virus strain with a titer of 109 PFU/l was procured from the Institute of Virology, Chinese Academy of Preventive Medicine. The titer used for infection was adjusted to 108 PFU/l by dilution with DMEM/F12 culture medium. ATRA was obtained from Chongqing Huapont Pharm Co. Ltd., China and the concentration used was 6 9 10-8 mol/l.

obtained from each mother and the protocol was approved by the institutional ethics committee. Mononuclear cells were isolated by density gradient centrifugation using lymphocytes separation medium. CD34? progenitor cells were enriched using CD34? cell isolation kit (Miltenyi Biotech Co., USA). Flow cytometry (Beckman–Coultcr Co., USA) was used to assess the purity of CD34?. The antibodies used FITC-conjugated anti-CD45? and PE-conjugated anti-CD34? (Immuotech Co., USA). The purity of CD34? cells was 93.4 ± 2.3%. Culture and Identification of CFU-E and CFU-TL The various study groups were designated as Control (blank), HCMV, ATRA ? HCMV, and ATRA groups. An optimized method developed in our laboratory was used for CFU-E/CFU-TL culture (Tables 1, 2). For identification of erythroid progenitor cells, Benzidine was added to the culture medium on days 3, 7, and 12. The morphology, size, and color of the cells were confirmed by microscopy. For identification of lymphocyte progenitor cells, cultured samples were collected at days 3, 7, and 12, stained by Wright–Giemsa staining method and the morphology, size of cells were confirmed by microscopy.

Collection of CD34? progenitor cells RNA extraction The umbilical cord blood samples, collected within 2–4 h after birth, were procured from Affiliated Hospital of Luzhou Medical College. Written informed consent was

Table 1 Lymphocyte progenitor cell culturing

Culture composition Fetal bovine serum (%) -4

2-Mercaptoethanol (10

mmol/l)

CD34? cells (105/ml)

HCMV human cytomegalovirus; ATRA all-trans retinoic acid; PHA-M phytohemagglutinin-M; PHA-TCM PHA-stimulated T cell conditioned medium

Table 2 Erythrocyte progenitor cell culturing

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Control

HCMV

ATRA

HCMV ? ATRA

30

30

30

30

5

5

5

5

3

3

3

3

HCMV (105 PFU/ml)

-

?

-

?

ATRA (6 9 10-8 mol/l)

-

-

?

?

DMEM/F12

?

?

?

?

PHA-TCM (%)

10

10

10

10

PHA-M (lg/ml)

10

10

10

10

Culture composition

Control

HCMV

ATRA

HCMV ? ATRA

Fetal bovine serum (%)

30

30

30

30

2-Mercaptoethanol (10-4 mmol/l)

5

5

5

5

5

HCMV human cytomegalovirus; ATRA all-trans retinoic acid

Total RNA was isolated from cells in different groups on days 3, 7, and 12 of culture using TRIzol reagent according

CD34? cells (10 /ml)

3

3

3

3

HCMV (105 PFU/ml)

-

?

-

?

ATRA (6 9 10-8 mol/l)

-

-

?

?

DMEM/F12

?

?

?

?

Erythropoietin (IU/ml)

10

10

10

10

Cell Biochem Biophys (2012) 63:133–141 Table 3 Target gene and TaqMan probe sequences

135

Primer F HOXB4 (CFU-TL)

50 -GCAAAGAGCCCGTCGTCT-30

GAPDH (CFU-TL)

50 -CCTCAAGATCATCAGCAAT-30

HOXB4 (CFU-E)

50 -GCA AAG AGC CCGTCG TCT-30

GAPDH (CFU-E)

50 -TGG GTG TGA ACC ACG AGA A-30

Primer R HOXB4 (CFU-TL)

50 -GAAATTCCTTCTCCAGCT-30

GAPDH (CFU-TL)

50 -CCATCCACAGTCTTCTGGGT-30

HOXB4 (CFU-E)

50 -GAA ATT CCT TCTCCA GCT-30

GAPDH (CFU-E)

50 -GGC ATG GAC TGT GGT CAT GA-30

TaqMan probe HOXB4 (CFU-TL)

50 -FAM-CGTGAACTTTGCGCATCCAGG-TAMRA-30

GAPDH (CFU-TL)

50 -FAM-ACCACAGTCCATGCCATCAC-TAMRA-30

HOXB4 (CFU-E) GAPDH (CFU-E)

50 -FAM-CGTGAACTTTGCGCATCC AGG-TAMRA-30 50 -FAM-ACCACAGTCCATGCCATCAC-TAMRA-30

to the manufacturer’s instructions, followed by detection using 10 g/l agarose gel electrophoresis and storage at -80°C until use for fluorescent real-time quantitative RQ-PCR assay. Real-time fluorescent quantitative-PCR Lymphocyte and erythroid progenitor cells were harvested on days 3, 7, and 12 for detecting HOXB4 mRNA expression by RQ-PCR. HOXB4 was amplified using 5 ll of cDNA synthesized from total RNA and the primers as described (Table 3). PCR products were detected by agarose gel electrophoresis and stored at -20°C until use. The cDNA templates were serially diluted tenfold and served as standards with presumed original copy number (104). A sample (5 ll) from each standard was added to a 30-ll reaction volume including 3 ll 109 buffer, 3 ll MgCl2 (25 mmol/l), 9 ll dNTPs (10 mmol/l), 1 ll dNTPs (10 mmol/l), 1 ll each of upstream and downstream primers (10 lmol/l), 1 ll TaqMan probes (10 lmol/l; Table 3), 0.3 ll Taq DNA polymerase, 14.8 ll DEPCH2O, and 5 ll cDNA template. PCR reaction was performed using the following reaction conditions: initial denaturation at 95°C for 60 s; followed by 45 cycles of denaturation at 95°C for 10 s; annealing at 55°C for 30 s; and extension at 72°C for 1 min. The specificity of PCR amplification was checked by melting curve analysis. The copy numbers of target genes were calculated from Ct values by using standard curves. The fold change in expression for each sample was calculated using 2-DDCt and the values for HOXB4 expression levels were represented as mean ± SD.

Western Blot In each group, 1 9 106 cells were collected at 3, 7, and 12 days, washed, lysed and proteins were extracted using standard method. The samples (20 ll aliquots) were stored at -80°C until use. The extracted proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), first at 80 V for 20 min then at 120 V for 50 min. After electrophoresis, the proteins were electroblotted to nitrocellulose membrane (at 200 mA for 80 min). The membranes were blocked in 10% skim milk solution at room temperature for 2 h and then treated with primary antibody (4°C, overnight). The membranes were washed thoroughly and then treated with secondary antibodies at room temperature for 1 h. X-ray films were exposed to nitrocellulose membranes in dark and developed. Radioactive bands were measured using image analysis software and HOXB4/Actin ratio was determined to measure relative amount of protein expression in the samples. Results regarding HOXB4 expression levels were represented as mean ± SD values from the data obtained from at least three independent experiments. Statistical analysis The data were calculated by a comparative threshold method using SPSS version 15.0. A pair wise comparison of mean values between groups (LSD method) was performed after detecting a significant difference by Homogeneity test for variance and two-way analysis of variance. Data were analyzed using Mann–Whitney rank sum test (except for ‘‘competitive (lymphomyeloid) repopulating

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units/CRU’’ assay). All P values of \0.05 were considered statistically significant.

Results Lymphocyte/erythroid progenitor’s identification by staining Lymphocyte and erythroid progenitor cells were stained using Wright’s Giemsa and Benzidine staining, respectively, and identified by fluorescence microscopy (Fig. 1).

Cell Biochem Biophys (2012) 63:133–141

were 138 and 141 bp, which were consistent with the expected fragments of HOXB4 and GAPDH, respectively. The expression of HOXB4 mRNA in CFU-E (Fig. 3) and CFU-TL (Fig. 4) precursors was time-dependent and showed an increase on days 3, 7, and 12 in culture. As shown in Table 4 and Figs. 3 and 4, expression levels of HOXB4 mRNA were significantly higher in ATRA group while the levels were lower in HCMV group as compared with those of control group (P \ 0.05). There was no significant difference between ATRA ? HCMV and control groups. Expression levels were significantly higher in ATRA ? HCMV group as compared with those of HCMV group (P \ 0.05).

HOXB4 mRNA expression HOXB4 protein expression HOXB4/GAPDH mRNA expression as determined by RT-PCR in CFU-E and CFU-TL precursors from different groups is shown in Fig. 2. The sizes of the amplified bands

Fig. 1 Lymphocyte (CFU-TL) and erythroid (CFU-E) progenitor staining. Lymphocyte and erythroid progenitor cells were stained using Wright’s Giemsa and Benzidine staining, respectively. Cells were identified by fluorescence microscopy. a CFU-TL colony (Day

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HOXB4/Actin protein expression in CFU-E and CFU-TL precursors at day 7 is shown in Figs. 5 and 6, respectively.

7, 9400); b CFU-E colony (Day 7, 9400); c lymphocytes of CFU-TL colony (Day 7, Wright’s Giemsa staining, 91,000); and d erythrocytes of CFU-E colony (Day 7, benzidine staining, 9400)

Cell Biochem Biophys (2012) 63:133–141

137

1.5 1.3 1.1

HCMV CONTROL ATRA+HCMV ATRA

0.9 0.7 0.5 0.3 0.1 Day 3

Day 7

Day 12

Fig. 3 Time-dependent expression of HOXB4 mRNA in CFU-E progenitors. Difference between days 3 and 7 in each group (P \ 0.05); difference between days 3 and 12 in each group (P \ 0.05); difference between days 7 and 12 in each group (P \ 0.05)

The expression of HOXB4 protein in CFU-TL (Fig. 7) and CFU-E (Fig. 8) precursors was time-dependent and showed an increase on days 3, 7, and 12 in culture. As

expression in CFU-TL precursors. (1) Control group; (2) HCMV group; (3) ATRA group; and (4) HCMV ? ATRA group

Expression level of HOXB4-mRNA

Expression of HOXB4-mRNA

Fig. 2 Agarose gel electropherogram of RNA extraction. a HOXB4 expression in CFU-E precursors; b HOXB4 expression in CFU-TL precursors; c GAPDH expression in CFU-E precursors; and d GAPDH

1.6 1.4 1.2

ATRA ATRA+HCMV CONTROL HCMV

1 0.8 0.6 0.4 0.2 0 Day 3

Day 7

Day 12

Fig. 4 Time-dependent expression of HOXB4 mRNA progenitors. Difference between days 3 and 7 in (P \ 0.05); difference between days 3 and 12 in (P \ 0.05); difference between days 7 and 12 in (P \ 0.05)

in CFU-TL each group each group each group

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Table 4 Expression of CFU-E and CFU-TL HOXB4 mRNA in normal cord blood among different groups ( x ± s, n = 10) Group

Level of HOXB4 mRNA expression Day 3

Day 7

Day 12

CFU-E

0.60 ± 0.04

0.81 ± 0.05b

0.91 ± 0.05c

CFU-TL

0.61 ± 0.03

0.85 ± 0.02b

1.22 ± 0.03c

0.32 ± 0.04a

0.58 ± 0.05a

0.76 ± 006a

a

0.39 ± 0.02

a

0.57 ± 0.03

0.82 ± 0.04a

0.88 ± 0.05a

0.99 ± 0.06a

1.19 ± 0.03a

0.82 ± 0.01a

1.22 ± 0.02a

1.57 ± 0.03a

0.63 ± 0.04d

0.77 ± .0.05d

0.87 ± 0.05d

Control

HCMV CFU-E CFU-TL ATRA CFU-E CFU-TL ATRA ? HCMV CFU-E

e

CFU-TL

e

0.68 ± 0.03

0.92 ± 0.03

1.18 ± 0.02e

CFU-E colony forming unit-erythroid; CFU-TL colony forming unit-T-lymphocyte a

P \ 0.05 vs. the same kind of cells in control group at the same time point

b

P \ 0.05 vs. the same kind of cells in control group on the 3rd day of culture

c

P \ 0.05 vs. the same kind of cells in control group on the 7th day of culture

d

P \ 0.05 and

e

P \ 0.01 vs. the same kind of cells in the ATRA group at the same time point

Fig. 5 HOXB4 protein expression in CFU-E at Day 7. (1) Control group; (2) HCMV group; (3) ATRA group; and (4) HCMV ? ATRA group. Difference between days 3 and 7 in each group (P \ 0.05); difference between days 3 and 12 in each group (P \ 0.05); difference between days 7 and 12 in each group (P \ 0.05)

shown in Table 5 and Figs. 7 and 8, expression levels of HOXB4 protein were significantly higher in ATRA group while the levels were lower in HCMV group as compared with those of control group (P \ 0.05). There was no significant difference between ATRA ? HCMV and control groups. Expression levels were significantly higher in ATRA ? HCMV group as compared with those of HCMV group (P \ 0.05).

Discussion The HCMV is a common pathogen responsible for asymptomatic and persistent infections in healthy individuals [8]. HCMV infection is a common disease and a hazard to human health, particularly in children. The

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Fig. 6 HOXB4 protein expression in CFU-TL at Day 7. (1) Control group; (2) HCMV group; (3) ATRA group; and (4) HCMV ? ATRA group. Difference between days 3 and 7 in each group (P \ 0.05); difference between days 3 and 12 in each group (P \ 0.05); difference between days 7 and 12 in each group (P \ 0.05)

hematopoietic system is one of the major organs involved in HCMV infection and hematopoietic progenitor cells are the potential sites for the viral occurrence [9]. One of the mechanisms of hematopoietic suppression caused by HCMV is related to its inhibitory effect on hematopoietic progenitor cells [10]. In this study, HOXB4 mRNA and protein expression was detected during the proliferation and differentiation of human cord blood hematopoietic stem cells into erythroid and lymphocyte progenitor cells caused by HCMV infection. The data show that HCMV markedly down-regulated HOXB4 expression which affected proliferation and differentiation of erythroid and lymphocyte progenitor cells. HOXB4 gene clusters are involved in the regulation of hematogenesis. It was shown [11] that forced expression of

Expression of HOXB4 protein

Cell Biochem Biophys (2012) 63:133–141 1.9 1.7 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0.1

139

HCMV CONTROL ATRA+HCMV ATRA

Day 3

Day 7

Day 12

Fig. 7 Time-dependent expression of HOXB4 protein progenitors. Difference between days 3 and 7 in (P \ 0.05); difference between days 3 and 12 in (P \ 0.05); difference between days 7 and 12 in (P \ 0.05)

in CFU-TL each group each group each group

Expression of HOXB4 protein

1.9 1.7 1.5 1.3 1.1

HCMV

0.9

CONTROL ATRA+HCMV

0.7

ATRA

0.5 0.3 0.1 Day 3

Day 7

Day 12

Fig. 8 Time-dependent expression of HOXB4 protein in CFU-E progenitors. Difference between days 3 and 7 in each group (P \ 0.05); difference between days 3 and 12 in each group (P \ 0.05); difference between days 7 and 12 in each group (P \ 0.05)

the transcription factor HoxB4 enhanced the self-renewal capacity of mouse bone marrow HSCs and conferred a long-term repopulating capacity to yolk sac and embryonic stem (ES) cell-derived hematopoietic precursors. HOXB4 activity was sustained at a high level during the early maturation stage of erythroid and lymphocyte cells which directly influenced the proliferation of progenitor cells at the late stage and accelerated the differentiation of hematopoietic stem cells [12]. HOXB4 was found to up-regulate transcription factor AP-1 and its subunits Jun-B and ra-1 that allowed for upregulation of cyclin D1 expression, suppression of cell cycle and acceleration of hematopoiesis [13]. Nuclear factor Y, thrombopoietin and AP-1 complex may participate in HOXB4 transcription [14]. Nuclear factor Y and thrombopoietin were found to up-regulate the expression of HOXB4 via the PKA and p38 MAPK signaling pathway, respectively [15]. In this study, HOXB4 mRNA and protein expression was observed in cord blood erythroid and lymphocyte progenitor cells in vitro which suggested that HOXB4 expression related with erythroid and lymphocyte hematopoiesis.

The mechanisms involved in the abnormal HOX gene expression caused by HCMV remain unclear. It was reported [7] that HCMV infection led to breaks in several chromosomal loci, commonly in the locus of 17q21–22 where the HOXB4 is located on chromosome 17. HOX gene expression is regulated by ATRA; HCMV major immediate-early promoter (HCMV MIEP) regulates the expression of ATRA receptor which may influence the proliferation and differentiation of hematopoietic stem cells [16]. The HCMV-MIEP may have an effect on the expression of HOXB genes due to the expression of HCMV genes induced by HCMV chromatin after infection [17, 18]; the effects on HCMV infection and p38 MAPK showed a chronological feature which was also cell typespecific [19]. In addition, thrombopoietin may upregulate expression of the HOXB4 via p38 MAPK resulting in abnormal hematopoietic function [20]. Therefore, HOXB4 expression induced by HCMV infection may be associated with MAPK signal transduction pathway. ATRA induces proliferation and differentiation in a variety of cells [21]. ATRA, at the concentration of 10-8 mol/l promotes the proliferation and reduces the apoptosis of hematopoietic stem cells. In this study, we found that ATRA upregulated the expression of HOXB4 during proliferation and committed differentiation of hematopoietic stem cells into erythroid and lymphocyte progenitor cells compared with control group induced by ATRA (6 9 10-8 mol/l). ATRA was found to induce expression of Hox genes depending mainly on its retinoic acid receptor (RAR) and retinene receptor that are present in cell nuclei. ATRA and the nuclear receptor in a dimeric form directly integrate with the ATRA-specific response element which causes activation or inhibition of gene transcription [22]. Allelic mutation of Hox genes is positively correlated with the phenotype and the degree of mutation is associated with the number of interference factors. For instance, ATRA at a low concentration can promote hematopoietic stem cell differentiation into erythroid cells while it can inhibit this process at a high concentration. ATRA was found to stimulate the differentiation of granulopoietic cells into terminal cells and influenced the differentiation of hematopoietic stem cells during the embryonic period of mammals [23]. ATRA at a concentration of 1 lmol/l was found to inhibit proliferation and differentiation of human hematopoietic stem cells in vitro [24]. The inhibition rate of human leukemia cell strains, such as HL-60, was 50% as induced by 3 9 10-10 to 10-9 mmol/l of ATRA while the proliferation rate was increased to 150% after treatment with 3 9 10-9 to 10-8 mmol/l of ATRA. Besides, HCMV-MIEA was found to increase the enhancer activity of interleukin-6 [25] and ATRA was found to inhibit interleukin-6 via its receptors [26]. Thus, it is speculated that the inhibitory effect of

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Table 5 HOXB4 protein expression in CFU-E and CFU-TL (x ± s, n = 10) Group

Level of HOXB4 protein expression Day 3

Day 7

Day 12

CFU-E

0.48 ± 0.04

0.81 ± 0.05b

1.18 ± 0.07c

CFU-TL

0.41 ± 0.01

0.65 ± 0.04b

1.17 ± 0.05c

0.34 ± 0.02a

0.68 ± 0.05a

0.86 ± 004a

a

0.29 ± 0.01

a

0.47 ± 0.02

0.87 ± 0.04a

0.71 ± 0.07a

1.09 ± 0.06a

1.39 ± 0.08a

0.82 ± 0.07a

1.35 ± 0.07a

1.62 ± 0.03a

0.58 ± 0.05d

0.87 ± .0.07d

1.23 ± 0.06d

Control

HCMV CFU-E CFU-TL ATRA CFU-E CFU-TL ATRA ? HCMV CFU-E

e

CFU-TL

e

0.68 ± 0.04

1.02 ± 0.06

1.38 ± 0.07e

CFU-E colony forming unit-erythroid; CFU-TL colony forming unit-T-lymphocyte a

P \ 0.05 vs. the same kind of cells in the control group at the same time point

b

P \ 0.05 vs. the same kind of cells in the control group on the 3rd day of culture

c

P \ 0.05 vs. the same kind of cells in the control group on the 7th day of culture

d

P \ 0.05 and

e

P \ 0.01 vs. the same kind of cells in the ATRA group at the same time point

ATRA on interleukin-6 via ATRA receptors may relieve HCMV infection to a certain degree. In this study, ATRA was used at a concentration of 6 9 10-8 mol/l based on the previous cell toxicity experiments as well as pertinent literature. The data show that ATRA at this concentration upregulates expression of the HOXB4 mRNA and proteins in normal hematopoietic progenitor cells. Additional studies are underway to determine the relationship between the concentration of ATRA and the expression of HOXB4 mRNA and proteins and to also investigate how the changes in ATRA concentrations influence the proliferation and differentiation of hematopoietic cells. In this study, we evaluated the hematopoietic inhibition caused by HCMV infection and the effect of HCMV/ ATRA on HOXB4 expression in vitro. Therefore, further studies will be required to determine how the HCMV inhibits hematopoiesis as well as how HCMV/ATRA regulates HOXB4 expression in vivo.

2.

3.

4.

5.

6.

7.

Acknowledgments We thank the Health Bureau of Sichuan Province for financial support (Grant No. 20060040). 8.

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