Genetic protection of repopulating hematopoietic cells with an improved MDR1-retrovirus allows administration of intensified chemotherapy following stem cell transplantation in mice

This study was undertaken to analyze the hematotoxicity of paclitaxel (Taxol) and to test whether transduction of repopulating hematopoietic cells with a retroviral vector (SF1m) expressing the human multidrug resistance 1 gene (MDR1) would permit dose intensification following bone marrow transplantation (BMT). While the regimen chosen (8 x 20 mg/kg i.p. within 12 days) produced a non-lethal, reversible hematotoxicity in mice with steady-state hematopoiesis, only 35.3% (6/17) of control mice survived when treated starting 14 days post BMT. In contrast, 83.3% (15/18) of mice transplanted with SF1m-transduced cells survived, owing to a significant protection against severe acute myelotoxicity (as determined by neutrophil counts, white and red blood cell counts and values for hemoglobin and hematocrit). After recovery from chemotherapy, an increase of myeloid cells that were resistant to colchicine and effluxed the fluorochrome Rhodamine 123 was observed in SF1m-mice, but not in controls. These results reveal that the lethal, dose-limiting hematotoxicity of an intensified post-transplantation chemotherapy with paclitaxel can be prevented by retroviral transfer of the MDR1 gene to a minor proportion of repopulating cells. Our mouse model, mimicking clinically achievable gene transfer rates, thus suggests that bone marrow chemoprotection may widen the therapeutic window and permit an earlier onset of post-transplantation chemotherapy.     

Hematopoietic recovery after autologous bone marrow transplantation in high-dose chemotherapy of cancer patients

In order to evaluate the possible utility of autologous bone marrow transplantation (ABMT), eighteen cancer patients were treated with high-dose combination chemotherapy supported by ABMT. We investigated CFU-GM/kg infused as well as bone marrow nucleated cells/kg to predict for hematopoietic recovery. Although hematopoietic recovery was not related to the nucleated cell number of the transfused bone marrow, CFU-GM infused related significantly to neutrophil recovery to 500/mm3 (correlation coefficient, r = -0.73, p less than 0.001) and platelet recovery to 50,000/mm3 (r = -0.69, p less than 0.001). Furthermore, a significant relationship of CFU-GM infused to the period of neutropenia below 500/mm3 was observed (r = -0.67, p less than 0.001). Taken together, CFU-GM dose can predict for neutrophil and platelet recovery, suggesting that ABMT can shorten the period of bone marrow suppression following high-dose chemotherapy.     

Dexamethasone as a chemoprotectant in cancer chemotherapy: hematoprotective effects and altered pharmacokinetics and tissue distribution of carboplatin and gemcitabine

Purpose Hematoprotective strategies may offer new approaches to prevent chemotherapy-induced hematotoxicity. The present study was undertaken to investigate the chemoprotective effects of dexamethasone and its optimal dose and the underlying mechanisms.Methods Lethal toxicity and hematotoxicity of carboplatin were compared in CD-1 mice with or without dexamethasone pretreatment. Plasma and tissue pharmacokinetics of carboplatin were determined in CD-1 mice. Carboplatin was quantified by HPLC. Gemcitabine was analyzed by radioactivity counting.Results Pretreatment with dexamethasone prevented lethal toxicity of carboplatin in a dose- and schedule-dependent manner. The best protective effects of dexamethasone pretreatment as measured by survival were observed at the dose level of 0.1 mg/mouse per day for 5 days (80% vs 10% in controls). In contrast, posttreatment with dexamethasone had no protective effects. Pretreatment with dexamethasone significantly prevented the decrease in granulocyte counts. To elucidate the mechanisms by which dexamethasone pretreatment reduces hematotoxicity, we examined the effects of dexamethasone pretreatment on the pharmacokinetics of carboplatin and gemcitabine in CD-1 mice. No significant differences in plasma pharmacokinetics of carboplatin or gemcitabine were observed between control and mice pretreated with dexamethasone. However, dexamethasone pretreatment significantly decreased carboplatin and gemcitabine uptake in spleen and bone marrow with significant decreases in AUC, T_1/2, and C_max, and an increase in CL.Conclusions To our knowledge, this is the first time that dexamethasone has been shown to significantly decrease host tissue uptake of chemotherapeutic agents, suggesting a mechanism responsible for the chemoprotective effects of dexamethasone. This study provides a basis for future study to evaluate dexamethasone as a chemoprotectant in cancer patients.This study was partly supported by funds for the Cancer Pharmacology Laboratory from UAB Comprehensive Cancer Center.     

Effect of the Cdk-inhibitor roscovitine on mouse hematopoietic progenitors in vivo and in vitro

Myelosuppression is one the most frequent side effects of chemotherapy. New agents that more selectively target cancer cells have been developed in attempt to improve the effects and to decrease the side effects of cancer treatment. Roscovitine is a purine analogue and cyclin-dependent kinase inhibitor. Several studies have shown its cytotoxic effect in cancer cell lines in vitro and in xenograft models in vivo. In this study, we investigated the effect of roscovitine on hematopoietic progenitors in vitro and in vivo in mice. The clonogenic capacity of hematopoietic progenitors was studied using burst-forming unit-erythroid (BFU-E), colony-forming unit granulocyte, macrophage (CFU-GM) and colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). In vitro, bone marrow cells were exposed to roscovitine (25–250 μM) in Iscove’s modified Dulbecco’s media for 4 h or to roscovitine (1–100 μM) in MethoCult media for 12 days. No effect on colony formation was observed after exposure to roscovitine for 4 h; however, concentration- and cell type-dependent effects were observed after 12 days. Roscovitine in concentration of 100 μM inhibited the growth of all types of colonies, while lower concentrations have shown differential effect on hematopoietic progenitors. The most sensitive were CFU-GEMM, followed by BFU-E and then CFU-GM. In vivo, mice were treated with single dose of roscovitine (50, 100 or 250 mg/kg) and the effect on bone marrow was studied on day 1, 3, 6, 9 or 12 after the treatment. In the second part of experiment, the mice were treated with roscovitine 350 mg/kg/day divided into two daily doses for 4 days. The bone marrow was examined on day 1 and 5 after the last dose of roscovitine. On day 1, BFU-E decreased to less than 50% of the controls (P = 0.019). No decrease in BFU-E formation was observed on day 5. No significant effect was observed on CFU-GM and CFU-GEMM growth after the treatment with multiple doses of roscovitine. Single doses of roscovitine or dimethylsulfoxide did not affect the colony formation. We also studied the distribution of roscovitine to the bone marrow after a dose of 50 mg/kg was administered intraperitoneally. Only 1.5% of the drug was detected in the bone marrow. Thus, the roscovitine effect on hematopoietic progenitors in bone marrow in vivo is only transient. One reason may be that only a small fraction of roscovitine reaches the bone marrow. Another explanation may be the short half-life observed for roscovitine that might not allow enough cell exposure to the drug. However, the toxicity of roscovitine to hematopoietic progenitors in vitro is within the same exposure range as cytotoxicity to cancer cells. Thus, precaution should be taken in clinical trials, especially when combinations with myelosuppressive cytostatics are used. Hairong Song and Marina Vita contributed equally to this work.     

Interleukin-1 alpha protects against the toxicity associated with combined radiation and drug therapy

A dose of total body irradiation sufficient to cause 70% mortality within 30 days (9.5 Gy) and maximally tolerated doses of either adriamycin (10 mg/kg) or cis-dichlorodiammine platinum (8 mg/kg) were administered to C57B1/6 mice and animal survival used as an index of toxicity. Whereas the nature of the toxicity resulting from the radiation alone was hematopoietic, the addition of either drug to the total body irradiation resulted in a pattern of animal death more consistent with that of gastrointestinal toxicity (100% dead within 7 days). However, if the radiation was delivered as a regional abdominal exposure, rather than total body, the gastrointestinal death observed following the combination of total body irradiation drug was not observed. The administration of 2.5 x 10(5) U IL-1 v24 hr prior to total body irradiation demonstrated significant protection against this dose of radiation (90% survival vs 30% survival). Similar protection was also observed when the IL-1 was administered 24 hr prior to the combination of total body irradiation with either drug. While these observations suggested that the IL-1 was protecting against gastrointestinal toxicity, subsequent studies demonstrated that IL-1, in addition to accelerating hematopoietic recovery following radiation insult, was equally effective in advancing the repopulation of the stem cell (CFU-GEMM) and progenitor cell (CFU-M and CFU-GM) compartments following drug treatment. Collectively, the data from these studies demonstrate that the lethal effects resulting from combined total body irradiation + drug treatment contain both a gastrointestinal and a hematopoietic component.     

胞苷脱氨酶基因对小鼠大剂量化疗的保护作用

Objective:To explore the feasibility of transfecting cytidine deaminase(CD)gene into mouse bone marrow cells in order to observe the drug resistance of high dose Ara-C and improve the tolerance of myelosuppression following combination chemotherapy.Methods:Human cytidine deaminase gene was transfected into mice bone marrow cells by retroviral vector.Resistant colony-forming unit granulocyte-macrophage(CFU-GM)assay was performed after the transfected mice bone marrow cells treated by the Ara-C.DNA was extracted from mice bone marrow cells.The drug resistant gene in mice bone marrow cells after transfection was detected by PCR.Results:Bone marrow cells of lhe donor mice cultured with lhe retroviral producer cells showed the drug resistant colonies and resistance to Ara-C,so did accept mice transplanted with the CD gene(CFU-GM of donor mice was 52%,X2=124.62,P＜0.01:accept mice was 54%,X2=126.26.P＜0.01,both compared with the contrast group).The animal survival rate was significantly higher in gene transfected group than that of the control(X2=7.42.P＜0.01).CD gene of transfected bone marrow cells was confirmed by PCR.Conclusion:CD gene can be transfected into bone marrow cells of mice efficiently and increase the drug resistance to Ara-C.     

Preclinical evaluations of therapies combining the vascular targeting agent combretastatin A-4 disodium phosphate and conventional anticancer therapies in the treatment of Kaposi's sarcoma

The antitumor efficacy of the vascular targeting agent combretastatin A-4 disodium phosphate (CA4DP) was evaluated in a xenograft model of Kaposi's sarcoma (KS) grown in athymic mice. Response to CA4DP alone or in combination with localized radiation treatment or systemic chemotherapy (cisplatin or vinblastine) was assessed using a clonogenic cell survival or tumor growth delay assay. Administering increasing doses of CA4DP to tumor-bearing mice resulted in a dose-dependent increase in tumor cell kill. CA4DP also enhanced the antitumor effects of radiation and chemotherapy ~ 10-100-fold. Although single doses of CA4DP as large as 300 mg/kg failed to alter tumor growth, the same total dose, administered as 3 fractions in 5 or 9 days, resulted in significant growth delay. Such repeated CA4DP exposures also significantly increased the response of KS xenografts to cisplatin. These findings suggest that CA4DP ought to be considered as a candidate agent for therapeutic evaluation in AIDS-KS patients.     

A phase I clinical, pharmacological, and biological trial of interleukin 6 plus granulocyte-colony stimulating factor after ifosfamide, carboplatin, and etoposide in children with recurrent/refractory solid tumors: enhanced hematological responses but a high incidence of grade III/IV constitutional toxicities.

A Phase I trial was conducted to determine the safety, biological activity, and hematopoietic recovery by the combination of interleukin 6 (IL-6) and granulocyte-colony stimulating factor (G-CSF) after myelosuppressive chemotherapy in children. Patients <22 years of age at diagnosis with either recurrent or refractory solid tumors received ifosfamide 1,800 mg/m2/day x 5 days, carboplatin 400 mg/m2/ day x 2 days, and etoposide 100 mg/m2/day x 5 days, followed by daily s.c. G-CSF (5 microg/kg/day) and IL-6 (2.5, 3.75, or 5.0 microg/kg/day). Pharmacokinetic, proinflammatory mediator levels, hematopoietic colony assays, and cytokine receptor expression studies were performed during course one. Nineteen patients were evaluable for toxicity and received IL-6 at doses of 2.5 (n = 8), 3.75 (n = 5), or 5.0 (n = 6) microg/kg/day. Dose-limiting constitutional toxicity occurred in two of six patients at 5.0 microg/kg/day, two of five patients at 3.75 microg/kg/day, and two of eight patients at 2.5 microg/kg/day. The maximum tolerated dose (MTD) exceeded the lowest dose tested. Because of lack of drug availability, an MTD was not established. The maximum concentration of IL-6 (2.5 microg/kg/day) was 0.799 +/- 1.055 ng/ml (mean +/- SD). During the first course, the median time to absolute neutrophil count > or = 1,000/mm3 and platelets > or = 100,000 mm3 was estimated at 19 and 23 days, respectively. Peripheral blood progenitor cells expressing receptors to IL-3, IL-6, and G-CSF increased significantly over baseline (P < 0.05). After the first dose of IL-6, IFN-gamma levels were abnormal in 13 patients, and IL-1beta levels were abnormal in 10 patients. IL-6 has a high incidence of constitutional toxicity and a lower MTD in children compared with adults. In vivo use of IL-6 in children after chemotherapy remains limited. However, IL-6 may be more optimally investigated in children under ex vivo conditions.     

Effect of chemotherapy on colony-forming unit-granulocyte macrophage from bone marrow and peripheral blood in patients with lung cancer.

The effects of chemotherapy on the growth of colony forming unit-granulocyte macrophage (CFU-GM) from the bone marrow (BM) and peripheral blood (PB) of 8 patients with lung cancer were studied by using recombinant interleukin-3, granulocyte macrophage-colony stimulating factor, and granulocyte-colony stimulating factor. After chemotherapy, the kinetics of CFU-GM depend on the type of chemotherapeutic regimens used: a rebound overshoot of CFU-GM in the PB was seen in the 4 patients treated with cisplatin plus etoposide (PVP) but not in the 4 patients treated with cisplatin plus mitomycin plus vindesine (CMV). In the BM, the recovery of CFU-GM was more retarded in patients treated with CMV relative to that seen in patients treated with PVP. The results obtained in simultaneous cultures revealed the differences between CMV and PVP in the kinetics of chemotherapy-induced myelosuppression. Determination of the optimal timing of in vivo use of hematopoietic growth factors after cytoreductive chemotherapy requires careful experimental design.     

Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice

The adoptive transfer of lymphokine-activated killer (LAK) cells combined with low dose interleukin 2 (IL-2) mediates the regression of established pulmonary metastases in mice and has efficacy in the treatment of human cancer. Systemic administration of high dose IL-2 alone can mediate tumor regression. Cortisone acetate (CA), 25-75 mg/kg, was administered daily to mice receiving high dose IL-2 for 10 days. CA significantly reduced the toxicity induced by IL-2; 38 of 48 mice receiving CA survived compared to 0 of 30 controls (P less than 0.0001). In addition, CA administration caused a decrease in IL-2-induced 125I-labeled albumin leakage in mouse organs. However, CA abrogated the in vivo antitumor effect of high dose IL-2, and to a lesser extent the therapeutic effect of exogenous LAK cells plus lower dose IL-2. Mice treated with 100,000 units of IL-2 showed 98, 63, and 33% reductions of pulmonary metastases in Hanks' balanced salt solution, 25 mg Ca/kg, and 75 mg Ca/kg groups, respectively; treatment with LAK and 7,500 units of IL-2 resulted in reductions of 94, 77, and 57% in these same groups. CA treatment of animals did not affect LAK generation, although the absolute number of LAK precursors was greatly reduced. These results show that although CA can reduce the toxic effect(s) of IL-2, it can be detrimental to successful immunotherapy using this approach.     

Phase I study of a combination chemotherapy of nedaplatin and cisplatin

A new platinum complex, nedaplatin, has been reported to be effective for both ovarian and cervical cancers. We designated a phase I dose-escalation study of a combination chemotherapy of nedaplatin and cisplatin to investigate the dose-limiting toxicity (DLT) and the maximum tolerated dose (MTD). Six patients, including two with advanced cervical cancer, three with ovarian clear cell adenocarcinoma and one with endometrial clear cell adenocarcinoma, were enrolled in this study. The doses of the two agents were escalated alternatively, i.e., a tandem method, from 40 to 80 mg/m2 by 20 mg/m2. Nedaplatin and cisplatin were administrated by intravenous drip infusion and repeated after an interval of at least 4 weeks, as a rule. The major toxicity observed was hematotoxicity. One of the 6 patients dropped out of this study because of severe hematotoxicity after 80 mg/m2 of nedaplatin and 60 mg/m2 of cisplatin were administered. With a dose of 80 mg/m2 nedaplatin and 80 mg/m2 cisplatin, severe neutropenia was found in all 6 patients, and thrombocytopenia and anemia were found in 1 patient, respectively. A slight hearing loss was detected by audiometry in 5 patients, but no one was inconvenienced in daily life. Mild nausea and vomiting were also observed in all 6 patients. In conclusion, the DLT of this combination therapy was hematotoxicity and the MTD was 80 mg/m2 for nedaplatin and 60 mg/m2 for cisplatin, respectively. Thus, 60 mg/m2 of nedaplatin and 60 mg/m2 of cisplatin may be recommended for combined administration.     

The small-molecule TNF-alpha modulator, UTL-5g, reduces side effects induced by cisplatin and enhances the therapeutic effect of cisplatin in vivo

We investigated a small-molecule modulator of tumor necrosis factor alpha (TNF-alpha), UTL-5g (also referred to as GBL-5g), as a potential chemoprotective agent against cisplatin-induced side effects including nephrotoxicity, hepatotoxicity and hematotoxicity. Pretreatment of UTL-5g i.p. in BDF1 mice reduced the levels of blood urea nitrogen (BUN) and creatinine induced by cisplatin treatment. The levels of both aspartate transaminase (AST) and alanine transaminase (ALT) in these animals were also reduced by UTL-5g. Pretreatment of UTL-5g did not significantly affect the number of white blood cells (WBC) under current experimental conditions, yet it markedly increased blood platelet counts by more than threefold. Therapeutic assessment in SCID mice inoculated with human HCT-15 tumor cells showed that UTL-5g did not attenuate the anti-tumor effect of cisplatin but increased the therapeutic efficacy of cisplatin. The LD50 of UTL-5g was determined to be > 2,000 mg/kg by an acute toxicity study. In summary, our studies showed that 1) UTL-5g significantly reduces nephrotoxicity and hepatotoxicity induced by cisplatin in mice, presumably by lowering the levels of TNF-alpha, 2) UTL-5g markedly increased blood platelet counts in mice and 3) UTL-5g treatment increased the therapeutic efficacy of cisplatin against HCT-15 cells inoculated in SCID mice.     

A comparison of the abilities of nitrobenzylthioinosine, dilazep, and dipyridamole to protect human hematopoietic cells from 7-deazaadenosine (tubercidin).

Nitrobenzylthioinosine, dilazep, and dipyridamole are potent inhibitors of equilibrative transport of nucleosides that may have pharmacological applications in modulating the therapeutic index of nucleoside antimetabolites used in cancer chemotherapy. We have compared the relative abilities of these inhibitors to reduce the toxicity of in vitro exposures to tubercidin against clonogenic progenitor cells of normal human bone marrow (CFU-GEMM, BFU-E, CFU-GM) and of two leukemic human cell lines (HL-60/C1, CCRF-CEM) that differ in their expression of transporter subtypes. Short (1-h) exposures to 1 microM tubercidin alone inhibited colony formation (a) of normal human hematopoietic progenitors (CFU-GEMM, BFU-E, CFU-GM) by 100%, and (b) of HL-60/C1 and CCRF-CEM cells by > 90%. Pretreatment (30 min) with nitrobenzylthioinosine, dilazep, or dipyridamole followed by simultaneous treatment (1 h) with these transport inhibitors during tubercidin exposures reduced toxicity against hematopoietic progenitors and cell lines. Greater reductions of toxicity were consistently seen with bone marrow progenitors and CCRF-CEM cells than with HL-60/C1 cells. For CFU-GEMM, BFU-E, and CFU-GM cells, reductions in tubercidin toxicity of 50-100% were achieved at these concentrations: > or = 0.1 microM (nitrobenzylthioinosine); > or = 0.1 microM (dilazep); and > or = 3.0 microM (dipyridamole). Pretreatment (30 min) followed by simultaneous treatment (1 h) with any of the transport inhibitors (> or = 0.1 microM) and 0.1 microM [3H]-tubercidin blocked the uptake of radioactivity completely in CCRF-CEM cells and only partially in HL-60/C1 cells. These effects, which were consistent with the nucleoside transport phenotypes of CCRF-CEM cells (inhibitor-sensitive) and HL-60/C1 cells (inhibitor-sensitive and inhibitor-resistant), suggested that protection was due to the inhibition of tubercidin uptake via equilibrative nucleoside transport system(s). Light-density mononuclear cells from human bone marrow, of which the clonogenic progenitors represented only a minor (< 0.01%) subpopulation, possessed far fewer nitrobenzylthioinosine-binding sites (2 x 10(4) sites/cell, Kd = 0.7 nM) than either HL-60/C1 cells (1.7 x 10(5) sites/cell, Kd = 0.9 nM) or CCRF-CEM cells (3.3 x 10(5) sites/cell, Kd = 0.5 nM). Initial rates of uptake of 1 microM [3H]adenosine (0-6 s, 20 degrees C) by human bone marrow mononuclear cells were reduced partially by 0.1 microM inhibitor (nitrobenzylthioinosine > dipyridamole > dilazep) and completely by 10 microM inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Circulating Hematopoietic Progenitors in Patients with Primary Lung Cancer

The levels of circulating hematopoietic progenitors were measured in 28 patients with primary lung cancer. The average numbers of progenitors per milliliter of blood were 33 (range 0–360) for colony-forming unit-granulocyte macrophage (CFU-GM), 23 (range 0–140) for burst-forming unit-erythrocyte (BFU-E), and 4 (range 0–50) for colony-forming unit-mixed lineages (CPU-mix). No significant influence of age, sex, histological type, or clinical stage of the tumor on the progenitor levels was detected. After cytoreductive chemotherapy of the patients by treatment with cisplatin plus etoposide, the cells showed 6- to 50-fold rebound overshoots, but no rebound was observed after treatment with cisplatin alone, cisplatin plus mitomycin C or cisplatin plus vindesine plus mitomycin C, or in 4 of 5 patients treated with cyclophosphamide plus adriamycin plus vincristine. Peripheral blood hematopoietic progenitors should be useful as an alternative source of stem cells for lung cancer patients treated with marrow ablative chemotherapy.     

Circulating hematopoietic progenitors in patients with primary lung cancer

The levels of circulating hematopoietic progenitors were measured in 28 patients with primary lung cancer. The average numbers of progenitors per milliliter of blood were 33 (range 0-360) for colony-forming unit-granulocyte macrophage (CFU-GM), 23 (range 0-140) for burst-forming unit-erythrocyte (BFU-E), and 4 (range 0-50) for colony-forming unit-mixed lineages (CFU-mix). No significant influence of age, sex, histological type, or clinical stage of the tumor on the progenitor levels was detected. After cytoreductive chemotherapy of the patients by treatment with cisplatin plus etoposide, the cells showed 6- to 50-fold rebound overshoots, but no rebound was observed after treatment with cisplatin alone, cisplatin plus mitomycin C or cisplatin plus vindesine plus mitomycin C, or in 4 of 5 patients treated with cyclophosphamide plus adriamycin plus vincristine. Peripheral blood hematopoietic progenitors should be useful as an alternative source of stem cells for lung cancer patients treated with marrow ablative chemotherapy.     

THE PRELIMINARY RESULTS OF TREATMENT OFADVANCED AND RECURRENT MALIGNANT LYMPHOMA BY BEAC REGIMEN SUPPORTED WITH AUTOLOGOUS HEMATOPOIETIC STEM CELLS TRANSPLANTATION

High dose chemotherapy with autologous hematopoietic stem cells transplantation (AHSCT) developed dramatically in the past decade, and great achievement has been made in the treatment of chemosensitive solid tumors, malignant lymphoma, multiple myeloma, and pediatric solid tumors. So far, high dose chemotherapy with autologous hematopoietic stem-cell support has been proposed as a potentially curative treatment for patients with recurrent, aggressive lymphoma that remains sensitive to further…     

联合应用成纤维细胞介导的G-CSF基因疗法及IL-2基因疗法对肿瘤的治疗作用

本实验观察了成纤维细胞介导的G-CSF基因疗法对大剂量化疗后造血功能损伤小鼠的恢复作用。结果发现:接受G-CSF基因治疗的实验小鼠外周血白细胞尤其是中性粒细胞降低程度明显减弱,恢复速度明显加快;并可明显促进血小板的恢复,但作用较缓;其脾脏和骨髓CFU-GM、CFU-MK、CFU-S水平显著地高于对照组,表明成纤维细胞介导的G-CSF基因疗法可显著降低大剂量化疗后造血损伤程度,并明显加速受损的造血功能的恢复。     

成纤维细胞介导的G-CSF基因疗法促进化疗后造血功能恢复的实验研究

利用RT-PCR方法克隆到包括有全部编码序列和部分5′、3′端非编码序列的人G-CSF cDNA,并通过核苷酸测序得到证实。将其定向克隆至逆转录病毒载体pLXSN,构建成人G-CSF的重组逆转录病毒表达载体pLGSN。体外经CRE和CRIP细胞的两次包装,病毒滴度达到了临床应用的水平(1.1×10~6CFU/ml)。hG-CSF逆转录病毒感染NIH3T3小鼠成纤维细胞后,分泌G-CSF达168U/ml,Southern分析表明hG-CSF基因已整合至NIH3T3-G-CSF细胞的基因组中,Northern和Western分析分别从mRNA和蛋白质水平证实了人G-CSF在NIH3T3-G-CSF细胞的表达。将NIH3T3-G-CSF细胞植入同系小鼠体内,能从血清中检测到持续表达的G-CSF活性。本研究为开展人G-CSF基因治疗奠定了基础。     

Dexamethasone exacerbates cytotoxic chemotherapy induced lethargy and weight loss in female tumor free mice

Cytotoxic chemotherapy can induce a systemic inflammatory response which has been proposed to be an underlying mechanism of cancer treatment related fatigue. Dexamethasone, a synthetic glucocorticoid that has potent anti-inflammatory effects, is incorporated into chemotherapy regimens to prevent chemotherapy-induced nausea and vomiting (CINV). The purpose of this study was to determine whether by suppressing cytotoxic chemotherapy-induced inflammation, dexamethasone could ameliorate chemotherapy induced fatigue/lethargy in tumor free mice. The effect of dexamethasone (DEX) on Cytoxan-Adriamycin (CA)-induced inflammation was assessed by measuring circulating levels of IL-1β, TNF-α, IL-6, GCSF, KC, and MCP-1 twenty-four-hours post CA injection. Decline in voluntary wheel running activity (VWRA) from baseline (used as a proxy for fatigue/lethargy), body weight and composition, and food intake were monitored in mice administered four cycles of CA every two weeks with or without DEX. CA increased circulating levels of IL-6, GCSF, KC, and MCP-1 and caused a rapid decline in VWRA and body weight immediately following CA-injection. Although the acute CA-induced decline in VWRA and body weight was not evident in mice administered CA + DEX, DEX alone had a suppressive effect on VWRA, and body weight continued to decline in mice administered both CA and DEX while it returned to baseline in CA-treated mice. CA or DEX alone had no long term impact on VWRA but DEX exacerbated lethargy and weight loss in CA-treated mice. Despite dampening the systemic inflammatory response to chemotherapy, dexamethasone failed to ameliorate acute or long term chemotherapy related fatigue/lethargy. Our pre-clinical findings suggest that supportive therapies like dexamethasone used to acutely control nausea and vomiting in cancer patients may actually contribute to overall symptom burden in cancer patients.     

卵巢癌患者血清中IGF1、IGF1R及AKT表达、血清CA125半衰期与耐药的关系

目的：检测IGF信号通路关键蛋白IGF1、IGF1R及AKT在顺铂耐药卵巢癌患者血清中的表达,检测血清CA125的动态变化,探讨与卵巢癌耐药患者预后的关系。方法：利用ATP-TCA法对40例卵巢癌标本进行药敏试验,其中16例对顺铂耐药,24例对顺铂敏感,ELISA法分别检测顺铂耐药和敏感组患者血清中IGF1、IGF1R及AKT的表达。采用微粒子捕捉免疫发光技术（MEIA）测定40例卵巢上皮癌患者治疗前、手术前及手术后7-14天、每疗程化疗后血清中CA125浓度,计算CA125半衰期,探讨与预后的关系。结果：IGF1、IGF1R及AKT在卵巢癌顺铂耐药组的表达显著高于敏感组（P值均为0.0001）,顺铂耐药患者CA125半衰期与敏感组患者相比有统计学意义。结论：IGF信号通路关键蛋白IGF1、IGF1R及AKT可能参与卵巢癌顺铂耐药,CA125半衰期联合IGF1、IGF1R及AKT检测可早期预测卵巢癌化疗耐药。