Introduction

Malignant tumors have been the common diseases detrimental to the health and life of human being. Chemotherapy is considered one of the most important treatment regimens.^{[1, 2]} To eliminate residual malignant cells and decrease drug resistance of tumor cells, a consolidation treatment should start with the shortest interval and higher dosage if possible.^{[3, 4]} However, myelosuppression is a dominant side-effect of the high dose chemotherapy, and the resulting delay in onset and dosage of consolidation chemotherapy is expected to reduce the anti-tumor effectivity. If the myelosuppression and multidrug resistance of tumor are prevented, the curative effect of chemotherapy will be improved obviously. In this study, the mdr1 gene was transferred into the bone marrow mononuclear cells which were autotransplanted into rabbits with VX2 hepatocarcinoma. In chemotherapy with doxorubicin, the relationships among myeloprotection, curative effects and chemotherapy dosage were observed. The results support that transferring of the mdr1 gene into bone marrow hematopoietic cells prevent over-dose chemotherapy-induced myelosuppression.

Methods

Reagents

Monoclonal antibody C219 of the mdr1 gene, ultrasen-sitive streptavidin-peroxidase immunohistochemistry kit and DAB stain were purchased from Maixin_Bio. Co., China. PCR reagents were purchased from Shanghai Biological Engineering Co., China. Cytokines were purchased from Sigama Chemical Co., USA. Doxorubicin, cyclophosphamide and daunorubicin were purchased from Zhejiang HISUN Drugs Limited Company, China.

Cells

The PA317-HaMDR1/A cell line (retroviral packaging cell line containing human mdr1 gene) was established and maintained in our laboratory. The cells were cultured in IMDM, supplemented with 10% (vol/vol) FCS and colchicine (90 ng/ml). Viral supernatants were harvested from confluent layers of these cells and concentrated by ultracentrifugation (40 000 rpm×2 hours), and the concentrated viral supernatants were filtered (0.4 µm) and kept frozen at  -70 ℃. The VX2 tumor cell line was purchased from Funabshi Farm Co., Japan.

Animals

A total of 40 New Zealand rabbits, which were 2.0-2.5 kg weight and 3-4 months old, were divided into 5 cohorts at random and 8 rabbits in each cohort, including cohort A (no chemotherapy), cohort B (ordinary dosage chemotherapy), cohort C (over dosage chemotherapy), cohort D (bone marrow mononuclear cells autotransplantation and over dosage chemotherapy), and cohort E (autotransplantation of bone marrow mononuclear cells transferred with the mdr1 gene and over dosage chemotherapy). This study was approved by the Institutional Ethics Committee of Chongqing University of Medical Sciences.

VX2 hepatocarcinoma rabbit model

Fixed on the operation table, each rabbit received general anesthesia and an incision of 2-cm long was made at the upper abdomen. After the right central lobe of the liver had been exposed, a 0.5-cm deep tunnel was made in the liver tissue and an 1 mm³ VX2 block was implanted into the tunnel. After bleeding was stopped, the incision was sutured. 

Separation and transduction of bone marrow mononuclear cells

After the rabbits of cohorts D and E were anesthetized, 4-6 ml of bone marrow was collected from femurs of each rabbit marrow puncture. The density gradient centrifugation method was used to purify the mononuclear cells of bone marrow. The cells (2.0-5.0×10⁵) were cultured in a humidified incubator at 37 ℃ and 5% CO₂ for 4 days with 3% glutamin, polyberene (200 µg/ml), erythropoietin (50 U/ml), interleukin-3 (2000 U/ml), interleukin-6 (100 ng/ml), stem cell factor (2000 ng/ml), granulocyte-macrophage colony stimulating factor (400 ng/ml), and concentrated viral supernatants. Cultured for 2 days, the fresh concentrated viral supernatants and cytokines were added.

Autotransplantation of bone marrow mononuclear cells

Twenty-four hours before the transplantation, the rabbits (except in cohort A) were given chemotherapy with a high dosage of cyclophosphamide (150 mg/kg) to leave the niches of bone marrow empty. After pre-treatment, bone marrow mononuclear cells were autotransplanted by two times at a 24-hour interval (cells counts 10⁶-10⁷/kg).

Monitoring therapeutic efficacy of tumor

All rabbits were examined regularly by ultrasound unit (Acuson Sequoia 512 Color Doppler Ultrasound Diagnostic Unit, USA). The echo and size of the tumor in the liver were monitored by bi-dimensional ultrasonograply, and the blood vessels in the tumor were observed by Color Doppler Energy Imaging (CDEI). Meanwhile, lymph nodes and organs in the abdominal cavity of each rabbit were monitored. After the rabbits were dead, the organs including the liver, lung, kidney, heart, and so on were enucleated and fixed in 10% buffered formalin, followed by pathological examination.

Monitoring bone marrow protection of the mdr1 gene

Peripheral blood was analyzed every week, and white blood cells were counted microscopically. Before and after transplantation, the chimerism and functional expression of the mdr1 gene in bone marrow mononuclear cells were detected.^[5] The chimerism of the mdr1 gene in transferred cells was detected by PCR using the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Weiterstadt, Germany), and primers mdr-sense strand (CCC ATC ATT GCA ATA GCA GG) and mdr-antisense strand (GTT CAA ACT TCT GCT CCT CA) amplified a 157bp fragment of the mdr1 cDNA.^[6] P-glycoprotein (P-gp), the product of the mdr1 gene in cell membrane and cytoplasm, was detected by streptavidin-peroxidase immunohistochemistry staining. In each smear, 200 cells were observerd and the positive cells were counted.^[7] Daunorubicin (DNR) efflux assay was performed to detect the efflux pump function of P-gp. Flow cytometry was performed with a FACS Calibur instrument (Becton Dickinson, San Jose, CA, USA), and 10 000 cells were detected and statistics were analyzed with the CellQuest software on a Power macintosh 7600 computer (Becton Dickinson).

Statistical analysis

The data were analyzed by the Chi-square test, Fisher exact probabilities analysis and analysis of variance with the SPSS10.0 statistical software. Median values and ranges were given.

Results

Chimerism and expression of the mdr1 gene in bone marrow mononuclear cells

After bone marrow mononuclear cells had been transfected for 4 days in vitro, the chimerism and expression of the mdr1 gene were detected. The specific fragment of 157bp was found in the cells which had been transfected for 2 or 4 days by PCR and was not found in untransfected cells (Fig. 1), indicating that the mdr1 gene had been integrated into the genome of the mononuclear cells. The expression and efflux pump function of P-gp were detected by SP immunohistochemistry staining and DNR efflux assay (Table 1). Comparing with the untransfected cells, the positive rate of P-gp expression in the transfected cells was increased (P<0.05) and the magnitude of DNR fluorescence was decreased obviously (P<0.05), which indicate that the  mdr1 gene could be functionally expressed in the transferred cells.

Chimerism and expression of the mdr1 gene in bone marrow

After bone marrow mononuclear cells had been autotransplanted for 1 month, the bone marrow mononuclear cells of rabbilts in the 5 cohorts were collected and detected. The specific fragment of 157bp was only detected in cells of cohort E by PCR. The positive rate of P-gp expression in cohort E was 8%, and this expression was hardly detected in cells of other cohorts (P<0.05). The positive rates of cells of DNR fluorescence were 29.27%, 45.01% and 56.96% in cohorts E, B and D respectively (Fig. 2), and the difference of DNR positive rates between cohorts E and B, and between cohorts E and D was significant as was shown by Chi-square test (P<0.05). So the mdr1 gene in the transfected mononuclear cells could be expressed functionally after autotransplantation.

Bone marrow protection of the mdr1 gene in over-dose chemotherapy

The white blood cell (WBC) counts of peripheral blood in each cohort were monitored every week (Table 2). After 2-fold-dose chemotherapy, all rabbits in cohorts C and D died of severe myelosuppression, and the WBC counts were lower than those in cohort E (P<0.05). After 3-fold-dose chemotherapy or chemotherapy had been over for 2 weeks, the WBC counts of rabbits in cohort E were higher than those in cohort B (P<0.05). Hence, transduction of bone marrow mononuclear cells with the mdr1 gene would permit dose intensification of chemotherapy agents following transplantation. 

Curative effects of over-dose chemotherapy

The size of liver tumor was monitored by ultrasonography (Table 3). Before chemotherapy, the size of hepatocarcinoma was almost the same in each cohort (P>0.05). After 2-fold or 3-fold-dose chemotherapy, the tumor size of rabbits in cohorts D and E was much smaller than that in cohorts B and A (P<0.05), indicating that hepatocarcinoma cells could be eradicated quickly and effectively by over-dose chemotherapy and the growth velocity of tumor was only decreased by normal dose chemotherapy. It was shown by CDEI that blood stream signals in tumors were asterism, short bandlike and branchlike, and the tumors were surrounded by big vessels and many branches implanted into them. The characteristics of blood stream (arterial or venous blood) and resistent index (RI) were detected by Spectrum Doppler Ultrasound. After over-dose chemotherapy, the vessels in tumors were decreased or extinct obviously in cohorts D and E. The blood stream of the tumor margin was sufficient in cohort B and very plentiful in cohort A, and tissues in the center of the tumors was colliquatively necrotic because of reduced blood stream.

Pathological examination showed curative effect and tumor metastasis in dead rabbits, and the survival time, healing rate and metastatic rate were recorded (Table 4). Comparied with those rabbits in other cohorts, showed a longer survival time (P<0.05), a higher healing rate (P<0.05) and a lower metastatic rate (P<0.05) in cohort E. The rabbits in cohorts A and B died of serious complications caused by widespread metastasis of malignant tumors. The rabbits in cohorts C and D died from multi-organ hemorrhage because of serious myelosuppression caused by over dosage chemotherapy. Thus, the survival time in the two cohorts was much shorter than that in cohorts A, B and E. These findings indicated that malignant tumor cells could be killed effectively, relapse and metastasis of tumors could be prevented, and the healing rate could be improved by myeloprotection and over dosage chemotherapy. Pathological examination revealed that most of the rabbits in cohort E died from serious cardiac toxicity induced by over-dose of adriamycin, and the deterioration of malignant tumor and myelosuppression were not the causes of death.

Discussion

Malignant tumors have been proven to be the systemic and heterogenetic disease. The metastasis and relapse of the tumors necessitate a therapeutic alliance including chemotherapy. Chemotherapeutic agents are injected into the vessels to eliminate the small metastatic sites all over the body as well as the original sites of the tumors. During chemotherapy, the MDR of tumor cells and myelosuppression are the main obstacles to healing of tumors, which are related to the mdr1 gene.^[8] It has been proven that the over expression of the mdr1 gene is attributable to the MDR of malignant tumors.^{[9, 10]} P-gp, the expression product of the mdr1 gene, is a drug efflux pump,^[11] and the concentration of chemotherapeutic agents in tumor cells is decreased because they are ejected by the P-gp pump^[12] with the development of MDR. However, normal bone marrow cells with little expression of P-gp are damaged by agents, causing serious myelosuppression. Hence the chemotherapy fails because of MDR of tumor cells and myelosuppression, and the conditions of patients will be deteriorated.

There is a dose-effect relationship between the dosage of chemotherapeutic agents and curative effects of malignant tumors. Animal experiments showed that the healing rate of the tumors would be decreased by 50% if the dosage of agents was reduced by 20%.^[8] In malignant tumors with a high growth index, the number of killed tumor cells may be increased 10-fold if the chemotherapeutic dose is increased by 2-fold. We conclude that malignant tumors would be cured if over-dose  chemotherapeutic regimen is used clinically. Bone marrow toxicity is a predominant side effect of many chemotherapeutic agents. Leucocytopenia or reduction of blood cells are caused by over-dose chemotherapeutic regiment, followed by serious complications like infection and hemorrhage.^[13] Finally, the dose of chemotherapeutic agents or therapeutic schedules are restricted or even ceased. The mechanism of MDR of malignant tumors gives some important clues that the foreign mdr1 gene can be transferred into bone marrow hematopoietic cells by advanced molecular biological techniques, which multidrug resistant to chemotherapeutic agents, and an over-dose chemotherapy schedule is feasible.^[14] The mdr1 gene was transferred into murine bone marrow hematopoietic cells through the retroviral vector SF1m, and the transferred cells are autotransplanted. In an experiment with paclitaxel, bone marrow protection of the mdr1 gene was observed.^[15] The WBC counts of the transferred mice were almost 5-fold higher than those of control mice, and levels of red blood cells, hematocrit and hemoglobin markedly improved.^[15] In another experiment,^[16] the mdr1 gene was transferred into the mobilized human peripheral blood progenitor cells (PBPC), with a transduction efficiency of 33%. The transferred PBPCs were subsequently transplanted into SCID mice, and 18% of the cells expressed the gene. An experiment also demonstrated that the bone marrow of SCID mice was protected after chemotherapy. In the above-mentioned experiments, reproducible virus was not detected in viral supernatant, bone marrow and peripheral blood of animals. Apparently, abnormal proliferation and function failure of bone marrow were not observed after transplantation with the mdr1 gene in mice. Based on successful animal experiments, clinical trials of the mdr1 gene transferring hematopoietic cells in patients with malignant tumors were permitted in USA.^[17-20] In phase Ⅰ clinical trials, the foreign mdr1 gene could be transferred into human CD34⁺ cells. And after autotransplantation, the transferring gene could be detected in bone marrow cells and peripheral blood cells of patients. However, the transduction efficiency and engraftment rate were low, and the bone marrow could not be protected effectively during chemotherapy. In viral supernatant, bone marrow and peripheral blood of the patients, reproducible virus was not detected. The results indicated that the transferring of the mdr1 gene into bone marrow hematopoietic cells is safe and reliable, but how to improve the transduction efficiency and engraftment rate is the focus of further study.

In this study, the mdr1 gene was transferred into the bone marrow mononuclear cells of rabbits with a transduction efficiency of 37% in vitro and the transferred cells were autotransplanted, with an engraftment rate of 8% in vivo. In the chemotherapy experiment, a normal dose of adriamycin could inhibit the rapid growth of VX2 hepatocarcinoma. However, bone marrow suppression occurred after 3-week chemotherapy, and diffusion and metastasis of the tumor happened finally. VX2 hepatocarcinoma cells could be killed rapidly and relapse and metastasis of the tumor could be prevented by over-dose chemotherapy. In this study, bone marrow toxicity was serious and fatal, and all rabbits died of multi-organ hemorrhage because of serious myelosuppression. The bone marrow mononuclear cells transferred with the mdr1 gene were transplanted, permitting dose intensification. Hence over-dose chemotherapy is advisable to kill hepatocarcinoma cells, prevent relapse and metastasis, and increase the survival time and healing rate of rabbits. But most of the rabbits died of cardiac insufficiency, acute pulmonary edema, and acute severe hepatitis. In future study, "suitable over-dose chemotherapeutic regimen" should be searched for according to dose of agents, bone marrow protection, curative effect of the tumor, and the damage to non-hematopoietic system.

The transduction of bone marrow hematopoietic cells with a retroviral vector expressing the human mdr1 gene would permit dose intensification following bone marrow transplantation, and malignant tumor could be cured by over-dose chemotherapy. The transplantation of bone marrow hematopoietic cells transferred with the mdr1 gene can mediate chemoprotection by over-dose chemotherapy and improve the curative effect of malignant tumors, thus providing a reference to clinical trials.

Acknowledgement

We are grateful for the technical assistance of the faculty of the Pediatrics Institute of Chongqing Children's Hospital of Chongqing University of Medical Sciences, Chongqing, China.

Funding: This study was supported by National Natural Science Foundation of China (No. 30330590).

Ethical approval: Not needed.

Contributors: JXQ proposed the study and wrote the first draft. LXJ analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. JXQ is the guarantor.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received September 5, 2005

Accepted after revision May 26, 2006