EFFECT ON BIOLOGICAL BEHAVIOR OF CHEMOTHERAPY-RESISTANT TUMOR CELLS BY HUMAN WILD-TYPE p53,GM-CSF AND B7-1 GENES VIA RECOMBINANTADENOVIRUS

Objective: To explore the effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes mediated via recombinant adenovirus. Methods: p53-abnormal KB-v200 (VCR resistant) and KB-s (VCR sensitive) cell lines were used as model tumor cells, which are resistant and sensitive to chemotherapeutic drugs respectively. After infected with recombinant adenovirus carrying human wild-type p53, GM-CSF and B7-1 genes, changes in biological behavior (including drug sensitivity) of these two kinds of gene-transduced cancer cells were observed. Results: Both of the cell lines were susceptible to adenovirus, all of three exogenous genes (p53, GM-CSF and B7-1) could be effectively expressed in these cell lines, their growth was suppressed, and apoptosis was induced. The drug-pumping-out function of Pgp glycoprotein on the cytomembrane of drug-resistant KB-v200 cells was markedly affected 48h after transfection of the recombinant adenovirus, revealed by increase of the amount of rhodamine 123 accumulation in the cells. The MTT assay also indicated the reversal of their sensitivity to VCR drugs.In vivo experiment in nude mice it was demonstrated reduction of tumorigenicity of the KB-v200 cells or KB-s cells infected with the recombinant adenovirus, and increase of their sensitivity to VCR. Conclusion: The clinical application of this recombinant adenovirus carrying agents might be more effective in treatment of tumors with multidrug resistance(MDR).

Experimental Study on the Antitumor Effect of Mouse B7-1 Gene

Summary: Mouse B7 1 cDNA was cloned by RT PCR from BALB/C mouse splenic cells and inserted into pcDNA3 to construct an eukaryotic expression vector. This constructor was named pCD mB7 1, in which the B7 1 cDNA was identified to be consistent with the data from other researchers. pCD mB7 1 plasmid was transfected into B16(F0) cells, and effective expression of mB7 1 in these tumor cells could be detected till the 6th month by RT PCR and RNA hybridization. Specific cytotoxity assay of lymphocytes was conducted after culturing with tumor cells and the results demonstrated that B16 cells transfected with B7 1 gene were more effective than B16 wt and B16 neo in inducing specific cytotoxity of lymphocytes against B16 wt cells. It is suggested that expression of B7 1 gene in tumor cells could enhance the immunogenicity and induce the effective antitumor immunity.

Antitumor Effects of the Fibroblasts Transfected TNF-α Gene and its Mutants

To compare the anti-tumor effects of transmembrane TNF-α(TM-TNF)and secreted TNF-α(S-TNF)in vivo,mouse fibroblasts NIH3T3 were transfected separately with three types of retrovirus containing wild type TNF-α(Wt-TNF),TM-TNF mutant(TM-TNFm),S-TNF mutant(S-TNFm).Southern blot,RT-PCR,FACS and bioassay were used to investigate TNF-αgene integration,expression and its biological activity.It was found that both fixed cells and supernatant of NIH3T3/Wt-TNF,the fixed cells of NIH3T3/TM-TNFm and the supernatant of NIH3T3/S-TNFm could express high level of TNF-αor its mutants and effectively kill H22 in vitro.The transfected NIH3T3 were separately injected into the mice at the sites of H22 tumor cell inoculation according to a ratio of 5∶1 or 1∶1(effector/target cells,E/T)after the third day of H22 challenge,respectively.At the E/T=5∶1,the NIH3T3/TM-TNFm induced the highest tumor regression,while NIH3T3/S-TNFm exerted the strongest tumor depressing effect at the E/T=1∶1 in vivo.No obvious side effects were noted throughout the course of treatment.The results suggest that both TM-TNF and S-TNF could cause tumor regression.The anti-tumor effect of TM-TNF would be more powerful and safe than that of S-TNF at the proper E/T ratio.

In vitro Study of Nucleostemin Gene as a Potential Therapeutic Target for Human Lung Carcinoma

Objective Nucleostemin （NS） is a GTP-conjugated protein located in the nucleoli of stem cells and some cancer cells, and maintains cell self-renewal. We aimed to evaluate NS as a potential target for lung carcinoma gene therapy by investigating NS gene expression and its effect on A549 cell proliferation. Methods NS mRNA and protein expression in A549, HepG2, SMMC-7721, HeLa, and U251 cells was analyzed by RT-PCR and western blotting following transfection of NS siRNAs and negative control siRNA （NC）. The effect on cell proliferation was also analyzed by MTF assays. Results NS mRNA and protein were both expressed in A549 cells and four other tumor cell lines; the relative expression levels were similar in all five cell lines. The three pairs of NS siRNA, either transfected alone or cotransfected into A549 cells, could effectively inhibit the expression of NS mRNA and protein. Moreover, the interference ratio showed an obvious concentration-dependent relationship. NS siRNA treatment resulted in significant inhibition of A549 cell proliferation by 35.7%. Conclusion NS gene was not only highly expressed but also played an important role in A549 cell proliferation. Thus, targeting of NS may be a promising novel strategy for the treatment of lung carcinoma.

Role for CD40L in the Therapy of Human Cancer

CD40L-CD40 interaction is central to the control of thymusdependent humoral immunity and cell mediated immune responses. CD40, a member of the tumor necrosis factor receptor （TNF- R） family, has been found on the surface of B lymphocytes, monocytes, hematopoietic progenitors, dendritic cells （DCs）, endothelial cells, epithelial cells and so on. Its natural ligand （CD40 ligand, CD40L）, CD154, a member of the tumor necrosis factor （TNF） family, is mainly expressed on activated CD4^＋ T lymphocytes. A direct growth-inhibitory effect can be found when ligated CD40 is on human breast, ovarian, cervical, bladder, non-small cell lung, and squamous epithelial carcinoma cells. This effect is related to the induction of cell cycle blockage and/or apoptosis. CD40L induces phenotypic and functional maturation of DCs, and can increase tumor immunogenicity through up-regulation of costimulatory molecular expression and cytokine production by epithelial cancer cells. As a result, CD40L can enhance tumor rejection immune responses. Furthermore, by means of a “bystander effect”, even CD40-negative tumor subsets can be eliminated by activated tumor-reactive cytotoxic T lymphocytes（CTL）. This review summarizes recent findings on CD40L recombinant protein and gene therapy-based tumor treatment approaches.

Preliminary Study on the Cooperation of IL-6 and mB7-1 in the Induction of Effective Antitumor Immunity In Vitro

To find a new way for gene therapy against tumors with weak immunogenicity, the effect of mB7 1 costimulation alone, or combined with IL 6, in inducing antitumor immunity in vitro was investigated. It was found that mB7 1 cDNA transfected B16 cells (B16 mB7 1) induced the expansion of effector lymphocytes and the generation of specific lytic activity more effectively than wild type B16 melanoma cells (B16 wt) or mock transfected B16 cells (B16 neo) did. ( P <0.01), IL 6 could effectively stimulate lymphocytes proliferation, but failed to enhance its cytotoxicity, while the combination of mB7 1 and IL 6 increased both lymphocyte proliferative response and T cell mediated cytotoxicity more significantly than B7 1 or IL 6 did alone ( P <0.01) . It was inferred that the costimulatory molecule B7 1 is required for the activation and proliferation of T lymphocytes; the expression of mB7 1 in tumor cells could increase their immunogenicity and induce effective antitumor immune response, and the combination of B7 1 and IL 6 could induce more effective antitumor immunity, indicating that cooperation of IL 6 and mB7 1 plays a role in T lymphocyte activation.

Replacement of the p16 gene in human ovarian cancer cells

Objective To study the inhibitory effects of retrovirus-mediated p16 gene on the human ovarian cancer cell line CAOV3.Methods The recombinant eukaryotic expression vector pDOR-p16 containing exogenous human wt-p16 cDNA and vector with neomycin resistance gene only were introduced into a CAOV3 cell line which does not express p16 endogenously by lipofectamine-mediated gene transfection. By using polymerase chain reaction amplification, mRNA in situ hybridization and immunocytochemistry, the clones obtained were tested for their efficiency of transfection and effects of vector expression. Their biologic behavior was observed further.Results Exogenous wt-p16 was transferred into CAOV3 cells successfully and permanent expression was obtained. The growth rate of the transfected CAOV3 cells in regular medium and soft agar was inhibited, and the tumorigenicity in nude mice showed that two of four mice failed to form tumors, and the others developed tumors 7 to 14 days later than mice of the contrast group. The percentage of phase G1 cells increased and that of phase S cells decreased. Under electron microscope, the ultrastructural changes of the cells revealed necrosis and growth retardation.Conclusions The p16 gene plays an important role in the generation and development of ovarian carcinoma. This study might provide experimental evidence for gene therapy in human ovarian cancer.

Spotlight on chimeric antigen receptor engineered T cell research and clinical trials in China

T cell mediated adoptive immune response has been characterized as the key to anti-tumor immunity. Scientists around the world including in China, have been trying to harness the power of T cells against tumors for decades. Recently, the biosynthetic chimeric antigen receptor engineered T cell(CAR-T) strategy was developed and exhibited encouraging clinical efficacy, especially in hematological malignancies. Chimeric antigen receptor research reports began in 2009 in China according to our Pub Med search results. Clinical trials have been ongoing in China since 2013 according to the trial registrations on clinicaltrials.gov.. After years of assiduous efforts, research and clinical scientists in China have made their own achievements in the CAR-T therapy field. In this review, we aim to highlight CAR-T research and clinical trials in China, to provide an informative reference for colleagues in the field.