Gene 542 (2014) 122–128

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High expression of heat shock protein 90 alpha and its significance in human acute leukemia cells Wen-Liang Tian a,1, Fei He b,1, Xue Fu a, Jun-Tang Lin c, Ping Tang a, Yu-Min Huang a, Rong Guo a,⁎, Ling Sun a,⁎ a b c

Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province 450052, China Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province 450052, China Key Laboratory for Medical Tissue Regeneration of Henan Province, Xinxiang Medical University, Xinxiang, Henan Province 453003, China

a r t i c l e

i n f o

Article history: Received 27 December 2013 Received in revised form 20 March 2014 Accepted 24 March 2014 Available online 25 March 2014 Keywords: Heat shock protein 90 Acute leukemia Expression

a b s t r a c t This study investigated the expression of heat shock protein 90 alpha (Hsp90α) in acute leukemia cells. The expression of Hsp90α was investigated in leukemia cell lines and human bone marrow mononuclear cells derived from acute leukemia patients and from healthy individuals using polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Compared with cells from healthy individuals, the expression of Hsp90α in the untreated patients was higher. Similarly high levels were observed in remission patients. Significantly higher expression levels were observed in all the tested cell lines, and in cells from refractory and relapsed patients. No obvious relationship was observed between the occurrence of graft versus host disease and the expression of Hsp90α. The untreated patients showing higher expression levels of Hsp90α had lower complete remission rates. During remission of untreated patients, the expression of Hsp90α decreased and reached the lowest level after transplantation, but the expression increased again before relapse. Hsp90α was highly expressed in leukemia cells. The expression level of Hsp90α was associated with leukemia prognosis. However, no obvious relationship was observed between the occurrence of graft versus host disease and the expression of Hsp90α. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Heat shock protein 90 (Hsp90) is a type of molecular chaperone that is found in various cell lines. Hsp90 was initially observed in cells exposed to elevated temperatures (Hartl and Hayer-Hartl, 2002; Young et al., 2004). Recent studies have shown that Hsp90 has an important function in the conformational maturation and stabilization of signaling proteins involved in cell growth and survival (Thomas et al., 2005; Whitesell and Lindquist, 2005). By regulating the function of various cancer proteins, Hsp90 participates in regulating tumor cell proliferation, survival, invasion, metastasis, angiogenesis, and other important processes (Didelot et al., 2007; Pearl et al., 2008). Inhibiting the

Abbreviations: Hsp90α, heat shock protein 90 alpha; Hsp90α, Hsp90 alpha; Hsp90β, Hsp90 beta; AL, acute leukemia; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ELISA, enzyme-linked immunosorbent assay; BMMCs, bone marrow mononuclear cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CR, complete remission; NR, non-remission; GVHD, graft versus host disease. ⁎ Corresponding authors at: Department of Hematology, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe East Road, Zhengzhou, Henan Province 450052, China. E-mail addresses: [email protected] (R. Guo), [email protected] (L. Sun). 1 Wen-Liang Tian and Fei He contributed equally as co-first authors.

http://dx.doi.org/10.1016/j.gene.2014.03.046 0378-1119/© 2014 Elsevier B.V. All rights reserved.

expression of Hsp90 can simultaneously regulate a wide variety of tumor signal pathways, and thus, it may have a critical function in tumor activity (Kamal et al., 2003, 2004; Lin et al., 2008; Solit et al., 2007). Depending on whether or not the protein contains rich regions of glutamine, human Hsp90 is divided into two categories: Hsp90 alpha (Hsp90α) and Hsp90 beta (Hsp90β). Hsp90α is important for the proliferation of tumor cells, whereas Hsp90β is associated with cell differentiation and structure building. Yano et al. (1996) found that Hsp90α mRNA expression is significantly higher in breast cancer tissue than in non-cancerous tissue, and that its expression is closely related to the expression of the nuclear antigen index of the proliferating cell, suggesting that high expression levels of Hsp90α are important for cell proliferation. Hsp90α gene shows high expression levels in pancreatic cancer and laryngeal cancer tissues (Gress et al., 1994). Significantly higher gene expression of Hsp90α was observed in the peripheral blood of patients with untreated acute leukemia (AL) compared with samples from healthy controls (Sedlackova et al., 2011; Xiao et al., 1996; Yufu et al., 1992). In previous studies, the expression of Hsp90α in patients with relapses, refractory disease, remission, or transplantation was not examined. Only the gene expression of Hsp90α was examined, and information on the protein expression of Hsp90α inside and outside the cells is lacking. We observed the differences in the expression of Hsp90α in different AL states to determine whether or not a correlation exists

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The following primers were used: (1) Hsp90α forward: 5 -ACCCAGACCC AAGACCAACCG-3 , reverse: 5 -ATTTGAAATGAGCTCTCTCAG-3 ; (2) GAPDH forward: 5 -TGCCCTCAACGACCACTTTG-3 , reverse: 5 -TCTC TCTTCCTCTTGTGCTCTTGC-3 . The primers were designed by the software primer3 (http://bioinfo.ut.ee/primer3-0.4.0/). For the real-time quantitative RT-PCR assay, the Hsp90α gene and the endogenous control GAPDH were amplified in different wells of a 96-well plate. Each well contained a reaction volume of 25 μl comprising 1 × Maxima SYBR qPCR Master Mix, 0.2 μM of the forward primer, 0.2 μM of the reverse primer, and 25 ng of cDNA. Each sample was analyzed in triplicate. The thermal profile used for the one-step real-time RT-PCR included denaturation at 95 °C for 10 min, followed by 40 cycles of PCR with denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. To eliminate the need for standard curves, the comparative Ct method (Kenneth and Thomas, 2001) was used to interpret the data. The difference (ΔCt) between the Ct values of Hsp90α and the endogenous control was calculated for each sample. The RNA isolated from the bone marrow of a randomly selected healthy control was used as the reference for each comparison. The comparative ΔΔCt calculation involved the determination of the difference between the ΔCt of each sample and the ΔCt of the reference. The ΔΔCt values were transformed to absolute values using the formula 2-ΔΔCt. Multiple negative water blanks (no template controls) were included in all the analyses. The samples were tested using a complete master mix without reverse transcriptase or primers to ensure that they were negative for DNA. No amplification was observed for these controls, indicating the specificity of the assays for the respective mRNAs.

between Hsp90α protein and gene expression, and to examine the association between Hsp90α and AL patient clinical outcomes. No similar reports have been published. 2. Patients and methods 2.1. Leukemia cell lines The cell lines used were K562 cells, a human chronic myeloid leukemia cell line (Center of Stem Cells, Zhengzhou University, China), Jurkat and molt-4 cells, a human acute lymphoblastic leukemia (ALL) cell line (Teaching And Research Section of Immunity, XinXiang Medical University, China), HL-60 and NB4 cells, and human acute myeloid leukemia (AML) cell line (Center of Stem Cells, Zhengzhou University, Zhengzhou, China). The five types of cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum and penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO2. 2.2. Subjects The protocol was approved by the Ethical Committee of the First Affiliated Hospital of Zhengzhou University, and informed consents were obtained from all the participants prior to testing. The untreated patients were sampled at the onset of the disease and prior to chemotherapy. The refractory and relapsed patients were sampled after diagnosis of refractory status or relapse but did not continue treatment. The remission patients were sampled after diagnosis of complete remission. Several cohorts of leukemia patients were included in the study. Patient data are shown in Table 1. All the cases were diagnosed at the Department of Hematology, The First Affiliated Hospital of Zhengzhou University. The healthy controls in the study included 14 healthy individuals (10 females and 4 males; age range, 14 years old to 52 years old; median age, 29 years old). The cohort was collected based on a suspicion of a hematological diagnosis that was ultimately determined as non-malignant or free of disease. Fresh bone marrow samples (4 ml) were collected from each subject. We centrifuged the samples, removed the supernatant, and stored the plasma samples in single-use tubes at −80 °C for further enzymelinked immunosorbent assay (ELISA) tests. We avoided multiple freeze– thaw cycles. The bone marrow mononuclear cells (BMMCs) were collected. A portion of the total BMMCs was used for isolation of total RNA, and another portion was used for isolation of total protein.

2.4. Western blot Protein from the BMMCs was isolated using a protein isolation kit (Sangon Biotech, Shanghai, China). Sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed with two gels simultaneously. Fifty micrograms of protein from the total cell lysate was concentrated on 5% SDS gels for 30 min at 100 V and separated on 10% SDS gels for 70 min at 120 V. The gels were transferred to polyvinylidenefluoride membranes (BBI, Canada) and blotted in parallel at 200 mA for 2 h. After blotting, the gels (Coomassie) and the membranes (Ponceau S) were stained to document successful blotting. The membranes were dipped in blocking buffer for 1 h and incubated with a polyclonal anti-hsp90α antibody (BBI, Canada) at 4 °C overnight. After washing three times with PBS-T, the membranes were incubated with a goat anti-rabbit secondary antibody (BBI) at 25 °C for 2 h. After washing three times with PBS-T, the bands were visualized on an X-ray film using the electrochemiluminescence method according to the manufacturer's instructions. The bands of the Hsp90α protein appeared at approximately 90 kDa, and the bands of the β-actin protein appeared at approximately 40 kDa. After scanning the X-ray film, image analysis was performed on a computer using the image processing software Image J.

2.3. Real-time quantitative PCR (qPCR) analysis Total RNA was extracted from cells using the RNeasy kit (Sangon Biotech, Shanghai, China). The cDNA was prepared by reverse transcription of the total RNA using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Beijing, China). The cDNA samples were stored at −80 °C until use. The experiment was performed using the Maxima SYBR qPCR Master Mix kit (Fermentas, Harrington, Canada) and the StepOne™ Real-Time PCR System (Applied Biosystem, Foster City, CA, USA). The system was applied according to the manufacturer's instructions. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as reference gene.

2.5. ELISA To perform the ELISA detection of plasma from the bone marrow, the plasma was centrifuged at 2400 ×g for 10 min. Precautions were taken

Table 1 Patients data in this study. Cohorts

Diagnosis

N

Females

Males

ALL

AML

Age range (years)

Median age (years)

First Second Third Fourth Fifth Sixth

De novo AL Remittent AL Refractory AL After HSCT Relapsed AL after chemotherapy Relapsed AL after HSCT

48 38 16 41 5 3

19 20 8 24 2 3

29 18 8 17 3 0

20 8 2 25 3 2

28 30 14 16 2 1

19–72 16–67 17–74 10–28 27–49 22–26

49 41 47 22 37 24

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to avoid hemolysis, and samples that have hemolyzed were not used. The plasma was transferred to a fresh tube for analysis in the assay. The concentrations of hsp90α were analyzed using ELISA (StressMarq, Victoria, Canada) according to the manufacturer's instructions. 2.6. Clinical observation All the untreated AL patients (except acute promyelocytic leukemia patients) underwent conventional chemotherapy regimens. AML patients were given DA (daunorubicin (DNR) at 45 mg/(m2·d), d1–3; cytarabine (Ara-C) at 100 mg/(m2·d), d1–7), IA (idarubicin at 12 mg/(m2·d), d1–3; Ara-C at 100 mg/(m2·d), d1–7), or TA (pirarubicin at 45 mg/(m2·d), d1–3; Ara-C at 100 mg/(m2·d), d1–7) chemotherapy regimen. ALL patients were given VDCLP (vincristine at 1.4 mg/(m2·d), d1,8,15,22; DNR at 45 mg/(m2·d), d1–3,d15–17; cyclophosphamide at 750 mg/(m2·d), d1,15; 2 L-asparaginase 6000 IU/(m ·d), d19–28; prednisone at 45 mg/(kg · d), d1–14, d15–17, start reduction to stop intake) chemotherapy regimen. Elderly patients were given reduced dosages on a case-by-case basis. After one course of chemotherapy, the relationship between the expression of Hsp90α and the remission status of the untreated AL patients was determined. The remission status was divided into two groups: complete remission (CR) and non-remission (NR) groups. The diagnosis and remission status were based on a standard reference (Zhang and Sheng, 2007). The diagnosis of graft versus host disease (GVHD) was based on the standard of the Seattle diagnosis and classification, which is commonly used in the clinical setting. We conducted a follow-up on specific patients to observe the disease outcome, and compared the relationship between the expression of Hsp90α and the outcome. 2.7. Statistical analysis The data were expressed as mean ± SD. Multiple comparisons were analyzed by ANOVA, and the comparison between the two groups was evaluated by Student–Newman–Keuls post hoc test. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Hsp90α gene expression detected by qPCR analysis The levels of Hsp90α gene expression in recurrent and refractory patients were similar, and thus, these patients were analyzed as a group. In our experiment, we used qPCR analysis to determine whether or not all of the tested cell lines, and the untreated, refractory, remittent, and relapsed leukemia patients had significantly higher expression of

Hsp90α gene compared with the transplanted leukemia patients and the healthy controls (P b 0.001). The relapsed and refractory patients showed significantly higher expression of Hsp90α in the qPCR analysis compared with the untreated leukemia patients (P b 0.001). The gene expression of Hsp90α in the untreated patients was higher than in the remittent patients, but no significant difference was found (P = 0.096) (Fig. 1). The remittent leukemia patients expressed higher Hsp90α levels compared with the transplanted patients and the healthy controls, and the difference between their levels was significant (P b 0.001). The transplanted patients and the healthy controls, the AML cell lines and the ALL cell lines, the untreated AML patients and the untreated ALL patients, and the patients with or without GVHD showed no significant differences in Hsp90α expression (P = 0.827, 0.377, 0.719, 0.626). 3.2. Hsp90α gene expression detected by Western blot analysis The levels of Hsp90α gene expression in the recurrent and refractory patients were similar, and thus, these patients were analyzed as a group. The results indicate that the untreated, refractory, recurrent, and remittent patients, as well as the leukemia cell lines, had significantly higher Hsp90α protein expression compared with the healthy controls (P b 0.001). The refractory and relapsed patients showed significantly increased expression of Hsp90α compared with the untreated and remittent leukemia patients (P b 0.05 and P b 0.001, respectively). The AML cell lines and the ALL cell lines, the untreated AML patients and the untreated ALL patients, the transplanted patients and the healthy controls, as well as the patients with GVHD and the patients without GVHD, showed no significant difference in Hsp90α expression (P = 0.571, 0.978, 0.360, 0.924), as shown in Fig. 2A and B. The results of the qPCR and the Western blot analysis were the same. 3.3. Hsp90α gene expression detected by ELISA analysis The levels of the Hsp90α gene expression in the recurrent and refractory patients were similar, and thus, these patients were analyzed as a group. The results indicate that all the patient cohorts, except the transplanted patients, had significantly higher Hsp90α protein expression compared with the healthy controls (P b 0.001). The refractory and relapsed patients showed significantly increased Hsp90α expression compared with the untreated and remittent leukemia patients (P b 0.05 and P b 0.001, respectively). The untreated ALL patients showed higher Hsp90α expression than the untreated AML patients, and the difference was significant (P = 0.012). The untreated leukemia patients had significantly higher Hsp90α protein expression than the remittent leukemia

Fig. 1. qPCR analysis of Hsp90α gene in the bone marrow samples and the leukemia cell lines. Comparisons of Hsp90α genes in different groups. The results are shown for all the groups of patients, including the untreated ALL patients (n = 20), the untreated AML patients (n = 28), the refractory AL patients (n = 16), the remittent AL patients (n = 38), the transplanted AL patients (n = 41), the relapsed AL patients (n = 5), the relapsed AL patients after transplantation (n = 3) and the healthy controls (n = 14).*P b 0.05 vs healthy controls, #P b 0.05 vs untreated AL.

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Fig. 2. Western blot analysis of Hsp90α protein in the bone marrow samples and the leukemia cell lines. A. Western blotting of Hsp90α protein. Lanes 1 and 2: untreated ALL patients; lanes 3 and 4: untreated AML patients; lanes 5 and 6: refractory AL patients; lanes 7 and 8: remittent AL patients; lanes 9 and 10: healthy controls; lanes 11 and 12 show: transplanted AL patients; lanes 13–17: relapsed AL patients; lanes 18–20: relapsed AL patients after transplantation. The leukemia cell lines are also shown above; B. Comparisons of Hsp90α proteins in different groups. The results are shown for all the groups of patients, including the untreated ALL patients (n = 19), the untreated AML patients (n = 20), the remittent AL patients (n = 13), the refractory AL patients (n = 13), the transplanted AL patients (n = 16), the relapsed AL patients (n = 5), the relapsed AL patients after transplantation (n = 3) and the healthy controls (n = 10). *P b 0.05 vs healthy controls, #P b 0.05 vs untreated AL.

patients (P = 0.003). The transplanted patients and the healthy controls, as well as the patients with and without GVHD, showed no significant difference in Hsp90α expression (P = 0.108, 0.352), as shown in Fig. 3. These results indicate that the Hsp90α protein expression levels in the cytoplasm and plasma were consistent. 3.4. Expression of Hsp90α and clinical observations After one course of chemotherapy, we collected information on the untreated AL patients who gained CR and NR. Four patients died during chemotherapy. After analyzing the remaining cases, we found that Hsp90α mRNA expression was significantly higher in patients who gained NR than in those who gained CR (P b 0.001). The CR rates were

lower in patients with higher Hsp90α expression. A similar result was found in the analysis of the Hsp90α protein expression (Fig. 4A, B, and C). We tracked and collected information on the clinical outcomes of 13 patients among the 29 who obtained CR after the first course of chemotherapy. Three patients relapsed and five patients stayed in remission with continuing chemotherapy. After a sufficient number of courses of chemotherapy, five patients underwent allogeneic hematopoietic stem cell transplantation (HSCT), and three of these five patients relapsed. In the patients that did not relapse, the expression of Hsp90α gradually declined as chemotherapy proceeded, and the expression of Hsp90α was lowest after allogeneic HSCT. Before relapse, the Hsp90α expression increased, and the highest level was obtained after relapse (Fig. 5A, B, and C). Other data are shown in Tables 2 to 4.

Fig. 3. ELISA analysis of Hsp90α protein in the bone marrow samples. The results are shown for all the groups of patients, including the untreated ALL patients (n = 15), the untreated AML patients (n = 24), the remittent AL patients (n = 36), the refractory AL patients (n = 14), the transplanted AL patients (n = 34), the relapsed AL patients (n = 5), the relapsed AL patients after transplantation (n = 3) and the healthy controls (n = 12). *P b 0.05 vs healthy controls, #P b 0.05 vs untreated AL.

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Fig. 4. A: qPCR analysis of Hsp90α gene in untreated, remittent (CR and NR), and refractory AL patients. The results are shown for all of the patients analyzed (n = 44), including the patients with CR (n = 29) and with NR (n = 15) after one course of chemotherapy. The hsp90α RNA expression levels in the BMMCs of the healthy controls (n = 14) and the refractory patients (n = 14) are shown. B: Western blot analysis of Hsp90α protein in untreated, remittent (CR and NR), and refractory AL patients. The results are shown for all the patients analyzed (n = 37), including patients with CR (n = 24) and with NR (n = 13) after one course of chemotherapy. The hsp90α protein expression levels in the BMMCs of the healthy controls (n = 10) and the refractory patients (n = 13) are shown. C: ELISA analysis of Hsp90α protein in untreated, remittent (CR and NR), and refractory AL patients. The results are shown for all the patients analyzed (n = 37), including the patients with CR (n = 24) and with NR (n = 13) after one course of chemotherapy. The hsp90α protein expression levels in the BMMCs of the healthy controls (n = 12) and the refractory patients (n = 14) are shown. *P b 0.05 vs healthy controls, #P b 0.05 vs CR.

4. Discussion Heat shock proteins have been identified in evolutionarily diverse organisms ranging from bacteria and plants to humans, and these proteins are categorized based on their molecular masses (Blake et al., 1995; Burrows et al., 2004). Four major families exist, including Hsp90. Hsp90 is ubiquitously expressed in the cytoplasm of normal cells, where it binds with many client proteins and is involved in homeostasis (Blake et al., 1995; Burrows et al., 2004; Hartl and Hayer-Hartl, 2002; Maloney and Workman, 2002; Sreedhar et al., 2004a; Takayama et al., 2003; Young et al., 2004). In normal cells, Hsp90 is expressed at relatively low levels and does not form complexes with other chaperone proteins (Kamal et al., 2003). Hsp90 is overexpressed in a wide variety of human tumors, which indicates that Hsp90 provides a survival advantage for tumor cells. Hsp90 also forms large complexes with other chaperone proteins in tumor cells to form the so-called multichaperone or superchaperone complexes (Isaacs et al., 2003; Jose et al., 2005; Kamal et al., 2003; Ochel and Gademan, 2002; Sedlackova et al., 2011; Sreedhar et al., 2004b). Previous studies report that an inhibitor of Hsp90 can inhibit the proliferation of tumor cells (Beliakoff et al.,

Fig. 5. A: qPCR analysis of Hsp90α gene in patients who maintained remission, relapsed after chemotherapy and relapsed after transplantation. B: Western blot analysis of Hsp90α protein in patients who maintained remission, relapsed after chemotherapy and relapsed after transplantation. C: ELISA analysis of Hsp90α protein in patients who maintained remission, relapsed after chemotherapy and relapsed after transplantation.

2003; Bonvini et al., 2002, 2004; Broemer et al., 2004; Chung et al., 2003; Fujiwara et al., 2004; Pittet et al., 2005; Xu et al., 2003; Yao et al., 2003). Hsp90 has two isoforms, Hsp90α and Hsp90β, which are 76% identical (Chung et al., 2003). Hsp90α has an important function in the proliferation of tumor cells. The expression of the hsp90α gene was higher in leukemia cell lines and in AL cells from leukemia patients compared with normal blood cells. The high expression of Hsp90α in leukemia cells may be associated with the active and indefinite proliferation of leukemia cells (Xiao et al., 1996; Yufu et al., 1992).

W.-L. Tian et al. / Gene 542 (2014) 122–128 Table 2 The qPCR results and the clinical features of all the untreated patients, shown for both case–control groups (patients who died in the course of chemotherapy are not included). Median hsp90α RNA expressiona (P25–P75) (qPCR)

a

Median hsp90α protein expressiona (P25–P75) (ELISA)

CR

NR

P

N

CR

NR

P value

25 19

16(1.0496 ± 0.9457) 13(0.9683 ± 0.9263)

9(1.9098 ± 0.8295) 6(1.9609 ± 0.8318)

b0.001 b0.001

Sex Male Female

23 17

15(2.6286 ± 1.3293) 12(2.6198 ± 1.1112)

8(3.5925 ± 1.1167) 5(3.5274 ± 1.4795)

b0.05 b0.05

Initial WBC (/μl) b10,000 27 ≥10,000 17

19(0.4825 ± 0.9344) 10(1.6552 ± 0.9346)

8(1.9487 ± 0.8121) 7(1.9242 ± 0.8339)

b0.001 b0.05

Initial WBC (/μl) b10,000 24 ≥10,000 16

17(2.2439 ± 1.2640) 10(2.9259 ± 1.5428)

7(3.4772 ± 1.4721) 6(3.7035 ± 1.8538)

b0.001 N0.05

Immunophenotype ALL 18 AML 26

12(1.0385 ± 0.9065) 17(1.0428 ± 0.9664)

6(1.8833 ± 0.8634) 9(1.9293 ± 0.8594)

b0.001 b0.001

Immunophenotype ALL 16 AML 24

11(2.6573 ± 1.3912) 16(2.6007 ± 1.4872)

5(3.5306 ± 1.0524) 8(3.5702 ± 1.2455)

b0.05 b0.05

Normalized to GAPDH.

A study by Sedlackova et al. (2011) preferentially focused on Hsp90α gene expression in leukemia cell lines. The diagnosis and prognosis of the patients were observed, but the number of samples was too small and the type of leukemia patients was limited to de novo AL. In the current study, we expanded the number of samples to observe whether or not correlation exists between the Hsp90α protein and gene expression and disease outcomes. We collected the bone marrow fluid of healthy controls and AL patients at different treatment periods. By qPCR, we analyzed the gene expression levels of Hsp90α and applied different primers to confirm the results of qPCR by RT-PCR analysis. By Western blot and ELISA, we detected the protein levels in cells and plasma. In the study of gene expression, the relapsed and refractory patients showed significantly higher Hsp90α expression compared with the remittent leukemia patients. The untreated and remittent leukemia patients had higher gene expression of Hsp90α compared with the transplanted leukemia patients and the healthy controls. The transplanted patients and the healthy controls, the AML cell lines and the ALL cell lines, as well as the untreated AML patients and the untreated ALL patients, showed no significant difference in the expression of Hsp90α. Our gene data and observations on the AL cell lines and the cells obtained from patients with untreated AL are consistent with the data of previous studies (Xiao et al., 1996; Yufu et al., 1992), in which higher Hsp90α mRNA levels in the AL cells were obtained from patients with untreated AL compared with healthy control samples. For protein expression, the Western blot and ELISA results were similar for some of the cases. The refractory, relapsed, untreated, and remittent leukemia patients had significantly higher Hsp90α protein expression compared with the healthy controls and the transplanted patients. The refractory and relapsed patients showed significantly increased expression of Hsp90α compared with the untreated and

Table 3 Western blotting results and the clinical features of all the untreated patients, shown for both case–control groups (patients who died in the course of chemotherapy are not included). Median hsp90α protein expressiona (P25–P75) (Western blotting) N

CR

NR

P

22 15

16(0.1432 ± 0.0623) 8(0.1514 ± 0.0.0527)

6(0.2930 ± 0.1102) 7(0.2795 ± 0.1428)

b0.001 b0.001

Initial WBC (/μl) b10,000 22 ≥10,000 15

15(0.1437 ± 0.0640) 9(0.1581 ± 0.0579)

7(0.2363 ± 0.2116) 6(0.3524 ± 0.1286)

b0.001 b0.001

Immunophenotype ALL 15 AML 22

10(0.1498 ± 0.0751) 14(0.1467 ± 0.0562)

5(0.2890 ± 0.1059) 8(0.2832 ± 0.1136)

b0.001 b0.001

Sex Male Female

a

Table 4 The ELISA results and the clinical features of all the untreated patients, shown for both case–control groups (patients who died in the course of chemotherapy are not included).

N Sex Male Female

127

Normalized to β-actin.

remittent leukemia patients. The protein expression level of the untreated leukemia patients was higher than in the remittent leukemia patients. In Western blot analysis, the untreated AML patients and the untreated ALL patients showed no significant differences in Hsp90α expression. In ELISA, untreated ALL patients showed significantly higher Hsp90α protein expression compared with the untreated AML patients. Considering the ELISA and Western blot results, the transplanted patients and the healthy controls showed no significant differences in Hsp90α expression. In the Western blot analysis, the five tested cell lines showed higher Hsp90α expression levels than the refractory patients, and the difference was significant. The AML cell lines and the ALL cell lines showed no significant difference in Hsp90α expression. We did not perform an ELISA on these cell lines. We analyzed the correlation between the Hsp90α protein and gene expression. Regardless of the gene expression and the protein expression levels, the results indicate that the relapsed and refractory patients showed significantly increased expressions of Hsp90α compared with the remittent leukemia patients. The remittent leukemia patients had higher levels of Hsp90α gene and protein expressions compared with the transplanted leukemia patients and the healthy controls. The transplanted patients and the healthy controls showed no significant difference in Hsp90α expression. We obtained different results using qPCR and RT-PCR analyses, but the Hsp90α gene and protein expression levels of the untreated leukemia patients were higher than in the remittent leukemia patients. All of the results of gene and protein levels were consistent, and the general results were consistent. We analyzed the correlation between the Hsp90α expression and the prognosis of the disease. All the untreated AL patients were observed after one course of conventional chemotherapy to analyze the information on patients who gained CR and NR. The CR rates were higher in the patients with lower Hsp90α expression than in those with a higher Hsp90α expression. The expression of the Hsp90α genes and proteins was associated with leukemia treatment because Hsp90α expression levels were lower in patients who received leukemia treatment. We tracked and collected information regarding the clinical outcomes of the patients who obtained CR after the first course of chemotherapy. The expression level of hsp90α was related with the level of remission. As chemotherapy proceeded, the expression of Hsp90α gradually declined and was lowest after allogeneic HSCT. The expression level became similar to that in healthy controls. Regardless of whether or not the patients underwent HSCT, the expression of Hsp90α increased with disease relapse to levels that were higher than at the start of the disease. The Hsp90α expression was higher in leukemia cells from patients with AL (except for the transplanted patients) compared with the healthy controls, especially in the refractory and the relapsed patients. By analyzing the correlation between Hsp90α expression and disease prognosis, we determined that high expression always correlates with low remission rates. The gene and protein expression levels were

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similar between the intracellular and extracellular regions. Detecting the expression level of Hsp90α can possibly assist with the prognosis of AL, and selective inhibition of Hsp90α may be a novel target for therapy in patients with leukemia. ELISA detection of Hsp90α appears to be a convenient method for predicting the prognosis of AL patients and for identifying leukemia patients who are most likely to benefit from Hsp90α inhibitor therapy. We determined that the five leukemia cell lines used in the study highly express Hsp90α, suggesting that these leukemia cell lines can be used in further studies on Hsp90α inhibitors. We do not know whether or not a correlation exists between Hsp90β expression and the occurrence and development of leukemia. Recent in vitro studies have shown that Hsp90 may be involved in the signaling pathways for cell proliferation, survival, and cellular adaptation. The molecular mechanisms include a variety of signaling pathways, such as PI3K, ERK1/2 and IKK (Gao et al., 2010; Jiang et al., 2011; Kurashina et al., 2009; Walsby et al., 2012, 2013; Okawa et al., 2009), but we do not know whether or not Hsp90α has the same function. Further studies are needed to investigate this phenomenon. In conclusion, Hsp90α gene was highly expressed in leukemia cells, and the gene and protein expression levels of Hsp90α were consistent. The expression level of Hsp90α was associated with leukemia prognosis. However, no obvious relationship was observed between the occurrence of GVHD and Hsp90α expression. Conflict of interest No competing financial interests exist. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81070445), Guangdong Natural Science Funds (No. 06020896), Guangdong Provincial Medical Science Research Funds (No. A2010021), China Scholarship Council (No. 2011841094), Henan Provincial Medical Scientific Research Fund (No. 2011020012), and Henan Provincial Health Technology Innovation Talent Project: Young and Middle-aged Technology Innovation Talent Project (No. 4112). References Beliakoff, J., Bagatell, R., Paine-Murrieta, G., Taylor, C.W., Lykkesfeldt, A.E., Whitesell, L., 2003. Hormone-refractory breast cancer remains sensitive to the antitumor activity of heat shock protein 90 inhibitors. Clinical Cancer Research 9, 4961–4971. Blake, M.J., Buckley, A.R., Zhang, M., Buckley, D.J., Lavoi, K.P., 1995. A novel heat shock response in prolactin-dependent Nb2 node lymphoma cells. Journal of Biological Chemistry 270, 29614–29620. Bonvini, P., Gastaldi, T., Falini, B., Rosolen, A., 2002. Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), a novel Hsp90-client tyrosine kinase: down-regulation of NPMALK expression and tyrosine phosphorylation in ALK(+) CD30(+) lymphoma cells by the Hsp90 antagonist 17-allylamino,17-demethoxygeldanamycin. Cancer Research 62, 1559–1566. Bonvini, P., Dalla Rosa, H., Vignes, N., Rosolen, A., 2004. Ubiquitination and proteasomal degradation of nucleophosmin-anaplastic lymphoma kinase induced by 17-allylaminodemethoxygeldanamycin: role of the co-chaperone carboxyl heat shock protein 70interacting protein. Cancer Research 64, 3256–3264. Broemer, M., Krappmann, D., Scheidereit, C., 2004. Requirement of Hsp90 for IkB kinase (IKK) biosynthesis and for constitutive and inducible IKK and NF-kB activation. Oncogene 23, 5378–5386. Burrows, F., Zhang, H., Kamal, A., 2004. Hsp90 activation and cell cycle regulation. Cell Cycle 3, 1530–1536. Chung, Y.L., et al., 2003. Magnetic resonance spectroscopic pharmacodynamic markers of the heat shock protein 90 inhibitor 17-allylamino, 17-demethoxydeldanamycin (17AAG) in human colon cancer models. Journal of the National Cancer Institute 95, 1624–1633. Didelot, C., et al., 2007. Anti-cancer therapeutic approaches based on intracellular and extracellular heat shock proteins. Current Medicinal Chemistry 14, 2839–2847. Fujiwara, H., et al., 2004. IC101 induces apoptosis by Akt dephosphorylation via an inhibition of heat shock protein 90-ATP binding activity accompanied by preventing the interaction with Akt in L1210 cells. Journal of Pharmacology and Experimental Therapeutics 310, 1288–1295.

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