Palladium/Silver Cocatalyzed Divergent Dimerization of 2H-Azirines for Regioselective Synthesis of Tetrasubstituted Pyrroles and Pyrimidines.

The unprecedented dimerizations of 2H-azirines have been developed under the catalysis of palladium and silver. Upon changing the reaction conditions, the fully aryl-substituted pyrrole and pyrimidine derivatives were furnished in moderate yields with regiospecificity, respectively. Control experiments uncovered distinct catalytic effects of two transition metals, and the proposed catalytic cycles plausibly rationalized the chemodivergence and regioselectivity.

Modular Synthesis of Tetrasubstituted Pyrroles via an Annulative Migration Reaction of Allenyl Ketones and p-Toluenesulfonylmethyl Isocyanide.

The metal-free cyclization of allenyl ketones and p-toluenesulfonylmethyl isocyanide (TosMIC), promoted by Cs2CO3, provides a convenient access to tetrasubstituted pyrroles in which an acyl group undergoes 1,2-migration. This tandem Michael addition/annulative migration synthetic strategy is general and high-yielding for various substituted allenyl ketones. Moreover, a phosphoryl or ester moiety is also a suitable functionality to enable such migration.

Inhibitory effect of the HPV-16mE6δ/mE7/TBhsp70δ vaccine on oral squamous cell carcinoma.

AIM: To evaluate the inhibitory effect of a recombinant human papillomavirus (HPV) fusion protein vaccine on oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: An animal model of OSCC was established using human peripheral blood lymphocyte reconstituted nonobese diabetic/severe combined immunodeficiency mice. HPV vaccine was subcutaneously injected into mice after tumor establishment. Tumors and spleens were measured, weighed and stained with hematoxylin and eosin. Lymphocyte subpopulations and cytotoxicity were analyzed with flow cytometric and cytotoxic T lymphocyte assay. RESULTS: The average weight and volume of tumors were significantly lower in the vaccine group than in the control group from day 27. Mice in both groups had high percentages of human CD3+ and CD3+CD8+ T lymphocytes. An elevated percentage of human CD3+CD16+56+ natural killer cells was found in the vaccine group. Moreover, vaccine increased the infiltration of human CD3 and UCHL-1+ cells in tumor tissues and enhanced cytotoxicity. CONCLUSIONS: The HPV fusion protein vaccine induces tumor cell death, lymphocyte infiltration and therefore suppresses tumor growth and protects against OSCC.

[Association of antigen processing machinery and HLA class I defects with clinicopathological outcome in laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the role of antigen-processing machinery (APM) component defects in HLA class I antigen down-regulation in laryngeal squamous cell carcinoma (SCC) and to assess the clinical significance of these defects. METHODS: Fifty-one formalin-fixed, paraffin-embedded SCC specimens were examined for the expressions of APM component transporter associated with antigen processing (TAP1) and low molecular weight polypeptide (LMP-7) and HLA class I antigen by immunohistochemistry. RESULTS: HLA class I antigens, TAP-1 and LMP-7 expressions were down-regulated in 56.9% (29/51), 39.2% (20/51) and 45.1% (23/51) of the tested specimens respectively, whereas HLA class I antigens, TAP-1 and LMP-7 expressions lost in 21.6% (11/51), 33.3% (17/51) and 27.5% (14/51) of the tested specimens respectively. TAP-1 and LMP-7 expressions were significantly correlated with HLA class I antigen expression (r=0.460, P<0.05 and r=0.685, P<0.05, respectively). HLA class I antigens down-regulation was significantly correlated with T stage (χ2=8.61, P<0.05). Both TAP-1 and LMP-7 down-regulations were significantly correlated with T stage (χ2 values were 9.72 and 8.97 respectively, P<0.05) and TNM stage (χ2 values were 9.18 and 7.70 respectively, P<0.05). TAP-1, LMP-7 and HLA class I antigen down-regulations were significantly associated with reduced patients' overall survival (P<0.05) and disease-free survival (P<0.05). Multivariate analysis showed lymph node metastasis, recurrence and HLA class I antigen down-regulation were unfavorable prognostic factors (P<0.05). CONCLUSIONS: Down-regulated expressions of HLA class I antigen and APM component TAP-1 and LMP-7 occur frequently in laryngeal squamous cell carcinoma, by which cancer cells could avoid immune surveillance, while HLA class I antigen down-regulation is a major contributing factor to tumour progression and mortality.

A tritherapy combination of a fusion protein vaccine with immune-modulating doses of sequential chemotherapies in an optimized regimen completely eradicates large tumors in mice.

Tumor-induced immunosuppression plays a critical role in both impeding tumor-specific immune responses and limiting the effects of cancer immunotherapy. Analyses of regulatory cells recruited during the growth of the E7-expressing tumor, TC-1, revealed a high percentage of regulatory T cells (Tregs) as well as myeloid-derived suppressor cells (MDSCs) in spleens and tumors. In this study, we proposed that treatment with immune-modulating doses of cyclophosphamide (CTX) and all-trans retinoic acid (ATRA) would result in a beneficial tumor microenvironment with the suppression of Tregs and MDSCs and, thus, enhance the effect of a human papillomavirus protein vaccine. Our results showed that CTX preconditioning and persistent ATRA treatment along with the vaccine achieved long-term survival and induced long-term memory responses. However, the effect of the antitumor response sharply declined when the tritherapy was initiated after the optimal therapeutic time. The more intensive regimen could rescue the effect of the tritherapy accompanied by the decreased percentage of Tregs and MDSCs in spleens and tumors. Besides, a favorable host environment was created by the reduced secretion of interleukin-10 and 6 and vascular endothelial growth factor (VEGF) in the tumor niche and decreased the expression of phosphorylation-signal transducer and activator of transcription 3 of TC-1 tumors. Our data shed light on the immune-modulating doses of sequential chemoimmunotherapeutic strategy targeting not only the tumor but also its microenvironment, which suggests a potential clinical benefit for the immunotherapy of HPV-associated malignancies.

PERP gene therapy attenuates lung cancer xenograft via inducing apoptosis and suppressing VEGF.

Inducing apoptosis is an attractive antitumor strategy. PERP is an apoptosis-associated target of p53, and its activation alone is sufficient to induce apoptotic pathway leading to cell death. We have previously demonstrated that overexpression of PERP in tumor cell lines with low intrinsic PERP activity suppressed cancer cell growth and enhanced sensitivity to chemotherapeutical agents. We further identified that PERP was present in surgical normal lung tissue, but absent in cancerous tissue of the same patient. Here, we sought to investigate the anti-tumor effects of PERP gene therapy in vivo. Then nude mice were transplanted with p53-mutanted Anip973 human lung cancer xenografts and treated with normal saline, pcDNA3.1 (vector) and pcDNA3.1-PERP, respectively. Successful transfection and robust expression of PERP was detected. Treatment with pcDNA3.1-PERP increased apoptosis and retarded growth in the xenografts, which contributed to a 55% decrease in tumor volume compared with controls. Furthermore, PERP gene therapy activated pro-apoptotic Caspase-3 cascade and upregulated the expression of the second mitochondria-derived activator of caspase (Smac) and human TNF-related apoptosis-inducing ligand (TRAIL), while suppressed vascular endothelial growth factor (VEGF) expression, indicating apoptosis and anti-angiogenesis are involved in the inhibitory effect of the PERP gene therapy. Taken together, our results suggest PERP gene therapy may supply an alternative strategy for lung adenocarcinoma management. Furthermore, Anip973 is a p53-mutanted cell line and the findings of this study provide reference value for other p53-mutanted cancers which is common among malignant tumors.

Combination of all-trans retinoic acid and a human papillomavirus therapeutic vaccine suppresses the number and function of immature myeloid cells and enhances antitumor immunity.

Despite advances in the development of human papillomavirus (HPV) prophylactic vaccines, little progress has been made in the field of therapeutic vaccines in recent years. In the present study, we found a significant accumulation of immature myeloid cells (ImC) in large TC-1 tumors and demonstrated that a HPV therapeutic vaccine restored antitumor immune responses with the correction of aberrant myeloid cell differentiation by all-trans retinoic acid (ATRA). Our study demonstrated that combining ATRA with vaccination not only decreased the number of Gr-1+ CD11b+ ImC, but for the first time also suppressed the function of Gr-1+ CD11b+ ImC with decreased expression of CD80. Furthermore, large numbers of CD11c+ CD80+, CD11c+ CD86+, and CD11c+ MHCII+ mature dendritic cells were recruited. The combination therapy generated significantly increased numbers of functional E7-specific T cells with elevated interferon- secretion and enhanced cytotoxic T-cell activity. These findings suggest potential clinical benefits for the combined use of ATRA and HPV therapeutic vaccines.

A novel therapeutic fusion protein vaccine by two different families of heat shock proteins linked with HPV16 E7 generates potent antitumor immunity and antiangiogenesis.

Human papillomaviruses (HPV), particularly HPV16, is considered a necessary cause of cervical and oral cancer. Thus, the development of a therapeutic vaccine against HPV is important for the control of cervical cancer. However, therapeutic vaccination has been limited by inadequate antigen-specific immune responses. Heat shock proteins (HSP), including calreticulin (CRT), HSP70 and gp96, have been shown to act as potent immunoadjuvant to enhance antigen-specific tumor immunity. Previous studies have shown that N domain CRT (NCRT) or C-terminal half of HSP70 (hsp) linked with HPV16 E7 are capable of inducing potent antigen-specific CTL activity in experimental animal models. Here we developed a recombinant NCRT/E7/hsp fusion protein to investigate the synergistic effects of NCRT and hsp for enhancing the potency of HPV16 E7 therapeutic vaccine and evaluated the immune responses induced by this fusion protein. Our results demonstrated that NCRT and hsp synergistically exhibited significant increases in E7-specific CD8(+) T cell responses and impressive antitumor effects against E7-expressing tumors. Furthermore, the NCRT/E7/hsp fusion protein also generates potent antiangiogenic effects. These results indicate that NCRT/E7/hsp fusion protein is a promising therapeutic vaccine for treatment of cervical cancer through a combination of antigen-specific immunotherapy and antiangiogenesis, with possible therapeutic potential in clinical settings.

[Advances in human papillomavirus therapeutic vaccine].

Human papillomavirus (HPV) infection is very common but with limited therapies available. Although the prophylactic vaccination will be promoted worldwide soon, it can only show its benefits decades later. For individuals who already have established infections and dysplasias, it has little efficacy. In contrast, the therapeutic vaccines bridge the temporal deficit by fighting against the established HPV infections and HPV-related diseases. HPV oncogenes may be delivered in viral and bacterial vectors, in peptides or protein, in nucleic acid form, or in cell-based vaccines. This review summarizes the clinical trials of HPV therapeutic vaccines under the way and the different preclinical research strategies that are under investigations.

Immunological protection against HPV16 E7-expressing human esophageal cancer cell challenge by a novel HPV16-E6/E7 fusion protein based-vaccine in a Hu-PBL-SCID mouse model.

Increasing evidence has suggested that infection with high-risk human papillomavirus (HPVs) is closely associated with esophageal squamous cell carcinoma (ESCC) in China. The E6 and E7 oncoproteins expressed in ESCC are considered as attractive tumor-specific antigen targets for immunotherapy. We have reported that the HPV16 mE6delta/mE7/TBhsp70delta fusion protein vaccination induced powerful anti-tumor immunity against TC-1 tumor cells in a C57BL/6 mouse model. In the present study, we further evaluate the protective efficacy of this fusion protein vaccine using an HPV E7-expressing human ESCC cell line (EC9706) and a Hu-PBL-SCID mouse model. We demonstrated that immunization with the fusion protein vaccine caused significant inhibition of tumor growth with the delay time to tumor detection (tests vs. controls, 16 d vs. 9 d, p<0.01) and much smaller tumor size (p<0.01) in vivo. The inhibitory rate was ca. 69.6%, and 25% of the fusion protein vaccinated-mice remained tumor free by the end of the experiment (42 d). Furthermore, the activated lymphocytes (CD8+) were capable of infiltrating into the tumor site, and much more apoptotic cells along with activation of caspase-3 were observed in the tumors from vaccinated-mice. Also, high expression levels of human IFN-gamma, TNF-alpha, granzyme B and perforin were detected in the tumors from vaccinated-mice. Therefore, we concluded that the HPV16 mE6delta/mE7/TBhsp70delta fusion protein vaccine is able to stimulate cellular-mediated immune response against E7-containing ESCC cells through CD8+-dependent CTL-induced apoptosis in Hu-PBL-SCID mice. These findings provide a scientific basis for HPV E7-expressing ESCC active immunotherapy.

Prophylactic, therapeutic and anti-metastatic effects of an HPV-16mE6Delta/mE7/TBhsp70Delta fusion protein vaccine in an animal model.

Human papillomaviruses (HPVs), particularly HPV-16, are not only causally linked to cervical cancers but also play an important role in the development of other cancers. The oncoproteins, E6 and E7, are consistently coexpressed in the majority of HPV-containing carcinomas and their metastatic lesions, and are critical to the induction and maintenance of malignant phenotype, and also can cause tumor metastasis. Therefore, E6 and E7 represent ideal tumor-specific antigens for the development of immunotherapy to prevent and treat HPV-associated cancers and their metastases. The powerful antigenic nature of Mycobacterium tuberculosis heat shock protein 70 (TBhsp70) is emphasized by evidence that mammals are capable of recognizing murine and human multiple B and T cell epitopes in this protein, and therefore allows it to be used as an adjuvant-free carrier to stimulate the immune response to a covalently linked fusion partner. In our present study, we developed a recombinant TBhsp70Delta protein expression vector that permits the production of other protein fused to TBhsp70Delta. A recombinant HPV-16mE6Delta/mE7/TBhsp70Delta fusion protein was expressed and purified, and immunization with the fusion protein in the absence of adjuvant was capable of providing strong protection to C57BL/6 mice against challenge and rechallenge with TC-1 cells, but not HPV negative Lewis lung cancer cells, and induced established TC-1 tumor regression and led to long-term survival. Consistent with the in vivo results, the fusion protein immunization in the absence of adjuvant induced cytolytic T lymphocytes recognized specifically TC-1 tumor cells in vitro. We also demonstrated that immunization with the fusion protein in the absence of adjuvant was effective in both preventing and treating TC-1 metastatic lesions in the lung metastasis model. In particular, immunization with the fusion protein caused regression of established lung metastatic lesions in 50% of immunized animals. This study represents an instance of tumor therapy with a TBhsp70Delta fusion protein and provides the scientific basis for the clinical application of the HPV16mE6Delta/mE7/TBhsp70Delta fusion protein in the treatment of HPV-associated cancers and their metastases.

Efficient expression of modified human papillomavirus 16 e6/e7 fusion protein and the antitumor efficacy in a mouse model.

Infection with human papillomavirus, particularly type 16 (HPV16), is highly associated with the development of cervical intraepithelial neoplasia and cervical cancer. The two early viral oncogenes, E6 and E7, are selectively retained and constitutively expressed in tumor cells and are therefore attractive immunotherapeutic targets. Thus a vaccine strategy based on recombinant HPV16 E6/E7 fusion protein represents an efficient approach against HPV16-associated tumors. Although the expression level of HPV16 E6/E7 fusion protein was presumed to be low, direct experimental proof in vivo was lacking. To enhance the expression level and investigate its antitumor efficacy in vivo, we constructed a modified HPV16 E6/E7 fusion gene with three point-mutations and expressed it in Escherichia coli. The encoded protein, denoted mE6(1-120)/mE7(1-60), comprises 120 N-terminus amino acids of E6 and 60 N-terminus amino acids of E7 plus a histine tag, was purified on an affinity column, and subsequently characterized by Western blotting. Immunization of mice with mE6(1-120)/mE7(1-60) completely protected them against subsequent challenge and rechallenge with TC-1 tumor cells expressing HPV16 E6 and E7 proteins. In the therapeutic experiments, most mice eliminated the preexisting tumors and had a long-term protection. Consistent with the results of in vivo experiments, the splenocytes from immunized mice elicited cytotoxic T lymphocytes and specifically lysed TC-1 cells in vitro. More importantly, the expression level of mE6(1-120)/mE7(1-60) was significantly improved, meeting the necessary quantity required for a vaccine clinical trial. In conclusion, these data provide a scientific basis for the use of modified mE6(1-120)/mE7(1-60) in future human trials.

[Isolation of E1A-related drug-sensitive new genes by suppression subtractive hybridization].

BACKGROUND & OBJECTIVE: It has been well demonstrated that E1A, as a tumor suppression gene, is capable of inhibiting the growth and metastasis of different tumors, and reversing the malignant phenotype. Particularly, the gene possesses the ability to greatly enhance the drug-sensitivity of tumor cells to several antitumor agents, and also increase the radio-sensitivity. However, the associated genes through which E1A can exert its antitumor functions still remain unknown. The aim of this study was to isolate E1A anticancer-related genes,which were differentially expressed in drug-sensitive tumor cells using suppression subtractive hybridization (SSH). METHODS: To construct SSH library of human lymph node metastasis tumor cells (LN686) using the mRNA from LN686 cells treated by E1A protein and the parental LN686 cells as tester and driver, respectively. Positive clones in the library were selected randomly, and dot blot was used for the analysis of expression pattern of the differentially expressed-gene fragments. The sequences of cDNA fragments were analyzed and compared with that in GenBank. The mRNA levels of the novel genes in tester and driver were determined by semi-quantitative RT-PCR analysis. RESULTS: The SSH library contained about 7000 positive clones. Random analysis of 384 clones with PCR demonstrated that 362 clones contained inserted fragments. The consequence of dot blot demonstrated that these genes were over-expressed in the tester compared to the driver significantly. The 362 clones were sequenced and BLAST analysis was conducted, 10 clones are shown to be novel ESTs, and were registered in GenBank. The mRNA levels of the seven novel genes were over-expressed in LN686 cells treated by E1A protein compared to those of parental LN686 cells by semi-quantitative RT-PCR analysis, and the difference of mRNA expression was approximately 3-8 times. CONCLUSION: Ten novel gene fragments were isolated by the SSH technology, and it provided the basis for further cloning their full- length genes and studying their functions.

[Expression of HPV16-E6 and E7 oncoproteins in squamous cell carcinoma tissues of esophageal cancer and non-cancer tissues].

BACKGROUND & OBJECTIVE: Evidences indicate that high-risk type human papillomavirus (HPV) are closely associated with the carcinogenesis, progression and transformation of several kinds of human tumors. This study was designed to determine the expression of HPV16- E6 and E7 oncoproteins in normal tissues, dysplasia tissues, and carcinoma tissues of patients with esophageal cancer and to investigate the biological significance of high-risk type HPV in the esophageal squamous cell carcinogenesis. METHODS: HPV16-E6 and E7 oncoproteins were determined using immunohistochemical staining in normal mucosa tissues (70 cases), dysplasia tissues (43 cases), and carcinoma tissues (18 cases). RESULTS: The positive rates of HPV16-E6 in the tissues of normal mucosa, dysplasia, and carcinoma of esophagus patients were 59.3%,88.4%,and 83.3%,respectively; the positive rates of HPV16-E7 protein were 62.1%, 90.7%, and 88.9%, respectively. The positive rates of HPV16-E6 and E7 in dysplasia and carcinoma of esophagus were significantly higher than those in normal mucosa (P< 0.05). Double expression of HPV16-E6 and E7 in normal mucosa was 25.7%, while in dysplasia and carcinoma were 88.3% and 83.3%,respectively. CONCLUSION: HPV16-E6 and E7 are highly associated with esophageal squamous cell carcinogenesis. And cooperation of HPV16-E6 and E7 may play an important role in genesis of esophageal squamous carcinoma.

[The anti-lung cancer activity of a modified HPV 16 type mE6Δ/mE7 fusion protein based vaccine in an experimental animal model].

BACKGROUND: To investigate the outgrowth inhibition of the HPV16-positive murine lung tumor induced by a modified HPV16 mE6Δ/mE7 recombinant fusion protein vaccine in vivo and provide a new clue for the further immunotherapy. METHODS: For prophylactic experiments, C57BL/6 mice were immunized with mE6Δ/mE7 fusion protein, and then inoculated with the TC-1 tumor cell, expressing HPV16 E6 and E7 viral proteins. On day 33 after inoculation, the tumor-free mice were re-challenged with a larger dose of TC-1 tumor cells. For therapeutic experiments, mice were vaccinated with mE6Δ/mE7 on days 3 and 14 after tumor cell inoculation. On day 60, the tumor-free mice were re-challenged with a larger dose of tumor cells. Tumor incidence and tumor volume of each group were calculated. MTT method was used to determine the proliferation of lymphocyte. RESULTS: In the prophylactic experiments, immunization with the mE6Δ/mE7 completely protected the mice against the tumor cell challenge and rechallenge, and all the mice remained tumor free during the 100 days' observation period. In contrast, all the mice in PBS and IFA-treated groups developed tumors within 6-12 days after the first tumor cell inoculation, and died of tumor burden within 30 days. In the therapeutic experiments, the tumor formation rates were 20%, 90% and 60% in vaccinated, PBS and IFA groups respectively. In the next larger dose of tumor cells rechallenge experiment, 87.5% of vaccinated mice still remained tumor free, but all the mice from either PBS or IFA group developed tumors with 4-6 days. In addition, the results of MTT indicated that the proliferation of lymphocytes from vaccinated mice was stronger than that from control group. CONCLUSIONS: The modified mE6Δ/mE7 can efficiently inhibit the growth of lung cancer in the animal model, indicating that mE6Δ/mE7 protein-based vaccine might show promise for the future clinical application.

[Effect of E1A gene on in vitro growth inhibition and radiochemosensitivity of lymph node metastasis cells of human head and neck squamous cell carcinoma].

BACKGROUND & OBJECTIVE: Adenovirus type 5 early region 1A (E1A) gene has been found to be a tumor suppression gene recently. The protein of E1A gene regulates the expression of many cellular genes positively or negatively, and possesses the activities of inducing differentiation of tumor cell, reversing of malignant phenotype, anti-carcinogenesis and anti-metastasis. Study of E1A protein on the treatment of lymph node metastasis of human head and neck squamous cell carcinoma (HNSCC) was not reported. This study was designed to investigate the growth inhibition and radiochemosensitivity of E1A gene on human lymph node metastasis cell line 686LN-1 derived from the patient with human tongue squamous cell carcinoma in vitro and its mechanism. METHODS: The pcDNA3-E1A recombinant plasmid, designed for high-level expression of E1A gene in a variety of eukaryotic cell lines,was transfected into 686LN-1 cells mediated by lipofectamine. To observe the growth inhibition of E1A gene on the cells, the growth curve and doubling time were investigated. Cells before and after transfection were treated with cisplatin, paclitaxel, bleomycin, and 5-fluorouracil (5-FU) for 24 hours or irradiation, respectively, then the changes of sensitivity were tested by MTT assay. The redistributions of cell cycle were analyzed by flow cytometry. Immunocytochemical staining was used to detect the expression of p53 and HER-2/neu. RESULTS: Compared with the vector-transfected cells (686LN-1-vect cells), the E1A-transfected cells (686LN-1-E1A cells) grew slowly, and the doubling time elongated (1.41-fold). 686LN-1-E1A cells showed distinct sensitivity to the anticancer drugs and irradiation. According to the IC(50) value, the sensitivity of 686LN-1-E1A cells increased approximately 8-fold to cisplatin, 20-fold to bleomycin, 10-fold to paclitaxel, 1-fold to irradiation compared with 686LN-1-vect cells. However, the sensitivity to 5-FU did not change. The cell cycle was dramatically arrested at G(2)/M phase in the 686LN-1-E1A cells. E1A gene remarkably suppressed the expression of HER-2/neu gene in 686LN-1-E1A cells. CONCLUSION: E1A gene can significantly inhibit the growth rate of lymph node metastasis cell line 686LN-1 of HNSCC. Moreover, it also slightly enhance the cell sensitivity to antitumor drugs and irradiation. These functions of E1A gene may be associated with its ability to suppress the HER-2/neu expression and to arrest the cell at G(2)/M phase.

[Selective myelo-protection by MDR1 and MnSOD genes regulated by a specific promoter].

OBJECTIVE: To study the specific protection of myeloid cells from chemotherapeutic agents and radiation. METHODS: The recombinant retroviral vectors containing MDR1 gene and MnSOD gene regulated by APN myeloid promoter were constructed and introduced into myeloblastic cell line KG1a and hepatoma cell line BEL7402. The resistance of the cells to antitumor drugs and radiation were analyzed by cell survival assay. In vivo, the murine bone marrow cells were isolated and infected by the retroviral particles, which were transplanted into recipient mouse treated with paclitaxel or X-ray. The murine white blood cell (WBC) was counted in order to assay the effects of MDR1 or MnSOD gene on hematopoiesis in the course of chemotherapy and radiotherapy. RESULTS: The resistance to chemotherapeutic agents such as cochicine, Vp-16, vincristine, doxorubcin and paclitaxel were elevated markedly by 10.6, 10.4, 11.2, 4.2 and 14.2 folds in KG1a cell line transduced with MDR1 gene. The resistance to radiation increased 3.7 folds at the dose of 10 Gy compared with parental cells in KGla cell line transduced with MnSOD gene derived by APN promoter. In contrast, the chemosensitivity and the radiosensitivity showed no significant change in BEL 7402 cell line transduced with MDR1 gene and MnSOD gene. In vivo, the WBC counts in the mouse introduced with MDR1 gene or MnSOD gene were higher than those in the control mouse (P < 0.01). CONCLUSION: The expression of MDR1 gene and MnSOD gene regulated by APN myeloid promoter is effective on myelo-specific protection without enhancing the resistance of tumor cells in vitro. The hematopoiesis can be reconstituted in vivo during anticancer drug or radiation treatment. This study may provide experimental evidence and new clues for myeloprotection of cancer patients being treated with chemotherapy and/or radiotherapy.

Expression of adenovirus type 5 E1A in the methylotrophic yeast Pachia pastoris and the inhibitory effect on S-180 tumor growth.

The human adenovirus type 5 (Ad5) early-region 1A (E1A) proteins have been shown to have strong tumor-suppressive activities in human tumor cells and to enhance the sensitivity of a variety of malignant tumors to apoptosis induced by ionizing radiation and chemotherapeutic agents. However, the inherent limitations of E1A gene therapy prevent its application, such as the efficiency of expression, precision of targeting, and toxicity of vector. This prompted us to construct an E1A expression vector (pPIC9/E1A) and express the E1A protein in the methylotrophic yeast Pichia pastoris. The E1A protein was purified using two steps of ion-exchange column chromatography on HiTrap Q and HiTrap SP. The analysis indicated that the E1A protein/liposome inhibited S-180 tumor growth and also rendered the S-180 tumor strongly susceptible to the anticancer drug bleomycin in vivo. Furthermore, tunnel assay clearly revealed that the mechanism was induction of cellular apoptosis. Importantly, the E1A protein overcame the limitations of gene therapy. Thus the E1A protein may be a useful therapeutic agent for some malignant tumors.