Postdoctoral Research Associate, Brown Cancer Center , University of Louisville , Louisville , KY , USA

Currently, as a postdoc research associate, I am working in the joined laboratories of Dr. Kelly K. McMasters, MD/PhD and Dr. H. Sam Zhou, PhD.

Dr. McMasters, the Sam and Lolita Weakley Professor of Surgical Oncology and director of the Division of Surgical Oncology, has overseen the development of the multi-million-dollar Surgical Oncology Clinical Research Center, including groundbreaking research directed by him in the treatment of melanoma, breast cancer and liver tumors. Gene therapy is another field in which Dr. McMasters is conducting oncology research, serving as the director of a National Institutes of Health-funded laboratory that examines potential genetic targets; Dr. McMasters has explored the use of viruses to deliver genes that kill cancer cells. In September 2002, he was awarded a $1 million, four-year grant from the NIH/NCI to continue the study of cancer gene therapy. Dr. Zhou received his doctorate in molecular biology from the University of Texas at Austin and currently is an assistant professor in the Department of Medicine and J. G. Brown Cancer Center at University of Louisville School of Medicine. He has excellent qualifications for the direction of proposed research, having published numerous research reports and reviews on engineered adenoviral vectors. During this time, as a key investigator, I’ve been conducting a list of very important projects, which are listed below:

Research Project #1 : Deletion of an endogenous promoter in the E1A regulatory region of adenovirus does not disturb viral replication despite reduced E1A expression

The activity of Adenovirus E1A proteins is to create a cellular environment for productive viral replication. There are a great of efforts to develop replication selective Adenovirus (RS Ad) vector in the hope that these vectors can destroy only cancer cells by using promoters that are activated in cancer cells to control E1A expression. Regulated Ad replication in target cells therefore could be a strategy for suppressing uncontrolled cell growth. Previous studies showed that recombinant Ad, in which expression of the E1A gene was designed to be regulated by heterologous tumor- or tissue-specific promoter(s), could kill the target tumors. However, several reports confirmed that the use of tumor-specific promoters in the context of Ad genome cannot always effectively activate the E1A gene. To study the effects of E1A levels on viral replication in cancer and normal cells, I have created three Ad vectors: Adhz63 containing WT E1A promoter, Adhz51 deleted entire E1A promoter and Adhz69 with heterologous CMV promoter in the E1A regulatory region. By using A549 human lung cancer cells and WI-38 normal lung cells, we found that the deletion of endogenous promoter or using the strong CMV promoter did not significantly disturb the viral replication of the oncolytic Ad, although they affected the E1A transcription and the partially deleted E1B-55K mutant viruses with altered E1A regulation could selectively replication in A549 lung cancer cells and spare WI-38 normal lung cells.

My data suggested that the virus replication was not always dependent on E1A protein expression in cancer cells and this gave the reasons why E1A-controling adenovirus by tumor promoter cannot always effectively activate the E1A gene. My discovery of this is very important for development of viral vectors to selectively kill off cancer cells clinically. These studies will be submitted for publication to the Journal of Cancer Gene Therapy, a highly regarded international journal.

Research Project #2 : Failing to induce E1A expression of EIB55K-deleting Adenoviruses cause less replication in Saos-2 cancer cells

A mutant adenovirus dl1520 with the deletion of E1B55K has been used in human cancer clinical trials because the virus selectively replicates in multiple cancer cells. Though it was reported that dl1520 can replicate in p53-defective cancer cells, the mechanism underlying dl1520 selective replication in cancer cells is still not well understood. Several groups reported that dl1520 could replicate in both p53-WT and p53-defective cells. To compare the replication in both Hep3B and Saos2 cells, I used dl1520 E1B-55K deleted Ad vectors, Adhz51 deleted entire E1A promoter and Adhz69 with heterologous CMV promoter in the E1A regulatory region to infect the cells, In Hep3B cells we could clearly detect cytopathic effect (CPEs) induced cell killing. In contrast, no CPEs were detected in infected Saos2 cells with Adhz51 and dl1520. Also, the infected Saos2 cells did show CPEs with Adhz69 with strong CMV E1A promoter. Western blot showed that no E1A expression with Adhz51 and dl1520. My data suggested that E1A failed to be induced in Saos2 cells caused the less replication with E1A endogenous promoter deleting and E1B55K deleting viruses. The results gave us better understanding the mechanism of E1B-deleted virus replication in Saos2 cancer cells which contains a C-terminal truncated RB protein. The truncated RB is somehow interrupted the function of Ad E1A proteins. Therefore, E1A cannot regulate cellular and viral gene expression and activate virus DNA replication. A manuscript based on these findings has been completed and submitted to the Journal of Gene Therapy, a very competitive peer-review journal of the highest quality in the field of cancer research.

Research Project #3 : Use of the probasin promoter ARR2PB to express E1A of adenoviral vector in Androgen receptor-positive prostate cancer cells for viral selective cytolysis of cancer cells

Transfection of reporter genes driven by ARR2PB was followed by a greater than 200-fold induction of reporter gene activity by dihydrotestosterone (DHT) in an androgen-dependent manner in androgen receptor (AR)-positive cells derived from prostatic epithelium. By using ARR2PB promoter, Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cell lines of non-prostatic origin or in AR-negative cell lines of either prostatic or non-prostatic origin. E1A products of adenovirus regulate host cell cycling programs and activate viral genes for efficiently virus propagation and infection. Controlling E1A selectively express in cancer cells may allow the virus replication only in cancer cells. I have created an Adenoviral vector with its E1A gene under the control of ARR2PB promoter with the goal of development of Ad vector that can selectively replicate within AR-positive prostate cancer cells but not in normal cells. These results will establish an unambiguous role for BAX in apoptotic processes in human prostate cancers and have implications for tumor-targeting killing strategies.

Research Project #4 : Adenovirus E1B proteins activate cell cycling gene expression and induce cell entering S phase for efficient propagation

Only approximately 20% of asynchronous HeLa cells infected with E1B 55-kDa mutant adenovirus support the virus to replicate and generate viral particles visible under transmission electron microscopy. By contrast, all cells infected with wild type virus produced progeny virus. In synchronously growing HeLa cells, 75% of the cells infected during S phase with the E1B mutant virus could replication, whereas only 10% of the cells infected during cell cycle G1 produced virus. However, wild type adenoviruses replicate in both S phase and G1 cells. This study demonstrated that E1B55K mutant virus selectively replicate in S-phase cells and is restricted in cell at G1 phase; therefore, E1B55K protein has the function to relieve this virus growth restriction at G1. Recently I have shown that WI38 cells infected with wild type virus activate cell cycling gene expression, including cyclin E, cdc25A and RAD21, with the advanced methods of cDNA microarray and real-time PCR. Whereas, virus deleted entire E1B gene cannot induce expression of cycling genes. My data suggest that E1B products may have important function in inducing cell from G1 enter S phase for efficient virus replication. In this project, we will answer several important questions such as whether cells infected with the E1B mutant virus synthesize viral DNA to levels of the wild type virus infection; to determine the time point when viral DNA started to replicate and how virus infection affects cell cycle etc.

Research Project #5 : Destroy Lung Cancer with Combined Effects of Apoptosis and Oncolysis

Enhanced selective replication of adenoviruses in cancer cells is one of the most promising approaches in terms of molecular cancer therapy. In this approach, cancer cells are selectively killed by amplified viruses, while normal cells are spared. Though genetically modified adenoviruses have been used in human clinical trials, the mechanism for virus selective replication in cancer cells has not yet well understood. Furthermore, more efficient viruses are required to achieve significant therapeutic effects. I have created more than 10 mutated adenoviruses, some of which cause complete destruction of lung and liver cancer cells. The goal of this application is to determine the key viral and cellular factors that allow mutated viruses to divide and multiply selectively in cancer cells. In this way, the cancer cells would be infected with a virus that would lead to cancer cell death, while sparing normal cells. I hope that, by understanding the manner in which the mutated virus destroys cancer cells, I will be able to design and develop even more effective gene therapy viruses which can be tested in animal models and , eventually, in patients. This will provide an advantage in our efforts to develop truly useful cancer molecular therapy treatments. I am quite optimistic that the promising preliminary data which I have generated predicts that this will be a valuable and novel approach to the treatment of cancer.

Research Project #6 : Prostate-specific expression of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) for targeted suicide gene therapy of prostate cancer

Prostate cancer is the most common primary cancer in men and the second leading cause of male cancer-related deaths in the USA . The most pressing problem is there are not effective treatments for this metastatic disease. Conditionally replicative adenoviral vector (CR-Ad) mediated a suicide gene is promising for prostate cancer therapy. The preclinical and clinical studies have demonstrated the safety and efficacy of this strategy for intratumor or local injection. However, it is limited for treatment of metastatic cancer, and the biggest concern is that they are also associated with a higher risk for adverse effects, especially in the light of the fact that there is no established effective therapy for serious, disseminated adenoviurs infection and subsequence organ toxicity. For safety and improved efficacy of this strategy, seeking powerful and less toxic suicide gene candidates and using tumor-restricted gene expression using transcriptional targeting techniques are two attractive approaches to reduce toxicity and potentially increase efficacy and safety of prostate cancer gene therapy. In this project, I will evaluate a novel suicide gene, multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK), for possible use in prostate cancer gene therapy. Our research strategy is to use prostate promoter ARR2PB control both Dm-dNK and the viral early region 1A (E1a) gene, which is important for virus replication; Controlling E1a selectively express in cancer cells may allow the virus replication only in androgen-positive prostate cancer cells and restrict expression of the Dm-dNK may cause the only prostate cancer cells could be killed upon administering anti-cancer prodrugs, sparing normal cells. This CR-Ad vector with the Dm-dNK prostate-targeting reconstitution models will provide an excellent system to study the efficacy and safety of the strategy; such system will be vitally important for evaluating the CR-Ad mediated a powerful suicide gene in treating metastatic prostate cancer, which is currently not available. The Dm-dNK gene might serve as both effective suicide gene for treatment of prostate cancer and anti-viral agent for prevention and treatment the possible disseminated adenovirus infection because of using replication-competent adenoviral vectors. Replication-competent vectors equipped with the fail-safe mechanism to abolish viral replication might improve safety significantly.

I propose to accomplish our goal by first studying the feasibility of this new strategy in vitro using contructed a replication-competent vector which harbors a Dm-dNK-IRES-E1a expression cassette driven by tumor-specific promoter. Once the positive in vitro data become available, I will use the techniques of mice carrying xenoplants to evaluate the efficacy and safety of the approach in mouse prostate cancer models.

There is a crucial need for seeking a safety and efficacy approach to treat prostate cancer, especially for metastatic cancer. My published and preliminary data recently strongly suggest that Dm-dNK can be expressed in some human cancer cells, that the enzyme retained its enzymatic activity. The evaluation of our new stretegy using selectively replicating Adenovirus-mediated Dm-dNK gene therapy for prostate cancer might develop a novel approach for metastatic cancer. The in vivo work will produce an animal model for testing such therapies. Several prodrugs being screened for this purpose in the tumor types that could be used in vivo at lower doses because of the high catalysis activity of Dm-dNK, if there is a research-based rationale supporting clinical trials. My work will address this need.

As the Principal Investigator, I have submitted this proposal to the Prostate Cancer Research Program of Department of Defense, USA .

Postdoctoral Fellow, Department of Molecular and Biomedical Pharmacology, University of Kentucky , Lexington , KY , USA ,

As a key investigator, my work was focused on study of the aryl hydrocarbon receptor (AHR) signaling pathway. The AHR is a basic helix-loop-helix transcription factor that binds toxic compounds such as dioxins. These compounds are pervasive environmental contaminants that are formed during combustion reactions and certain manufacturing processes. The endogenous ligand for the AHR is not known. Genes that are regulated by the AHR include cytochrome P4501A1. One of my projects focuses on DNA binding of the AHR and its DNA binding partner the AHR nuclear translocator (ARNT). I am interested in how these proteins contact DNA and have determined the amino acids that contact the DNA recognition site. In addition, we are interested in determining whether other proteins such as p27kip may play a role in gene activation by the AHR and ARNT. I have found that transcription factor IIB interacts with the AHR and increases its ability to bind DNA. A second project involves determining the physiological role of the AHR. An understanding of what genes the AHR regulates under normal conditions will help us understand how compounds like dioxin may cause cancer. Towards this end, we are using cultured human keratinocytes to identify the gene pathways involved in control of proliferation and differentiation that are targeted by dioxin.

I had successfully characterized that in addition to the function of P27kip-1 in Carcinogenesis of dioxins, by generating two 21 bp dsRNA oligonucleotides of Arnt, which is a partner molecule of AhR and an important member of a structurally related gene family with characteristic structural motifs designated as bHLH (basic helix–loop–helix) and PAS (Per, Arnt/AhR, Sim homology), the Arnt expression had been significantly suppressed. My studies demonstrated that siRNA can be used to downregulate the expression of Arnt genes expressed in human cancer cell line. I also showed that siRNA-induced suppression of Arnt expression results in significant inhibition of TCDD-induced expression of CYP1A1 in HepG2 cancer cells. These findings suggest that delivery of siRNA directed against Arnt might be a powerful approach which decreases the toxic carcinogenesis effect of TCDD. The result is encouraging and is of great impact on Chemical Carcinogenesis and Toxicology.

These unique and significant discoveries not only are important to a further understanding of the toxic carcinogenesis effect of TCDD, but also have a fundamental impact on clinical application and treatment of cancer. These exciting results will be published in peer-reviewed journals and presented at an international conference.

In addition to the scientific discoveries, I improved some crucial cancer research techniques. For example, I independently established and improved a Methodology of Reverse Transcription Polymerase Chain Reaction (RT-PCR). RT-PCR is a very sensitive technique to measure and to compare mRNA levels among samples. My new methodology is simple and accurate as indicated by equal amplification efficiency throughout PCR cycling. It is also easily implemented for many existing protocols.

Ph.D. student, Both in: China Medical University , Shenyang , P. R. China. Karolinska Institute, Stockholm , Sweden .

I was admitted to a Ph.D. program in 1998 after passing a very competitive National Examination. During my Ph.D. period, I became involved in the research laboratory of Prof. Renxuan, Guo M.D. Ph.D., focusing on the Gene Therapy of Cancer. I applied molecular biology and cell biology techniques to study the immunological characteristics of cancers and develop novel effective strategies of cancer treatment. The Ph.D. studies were carried out in collaboration with Prof. Anna karlsson and Dr. Magnus Johansson at the Department of Microbiology, Pathology and Immunology, Huddinge University Hospital, Karolinska Institute, Stockholm Sweden from 1999, and I was registered as a Ph.D. student at Karolinska Institute after passing qualified examination in Stockholm in 2000. During my Ph.D. period, I participated in various research projects. One of the important projects is “Evaluation of the deoxyribonucleoside kinase of Drosophila Melanogaster (Dm-dNK) as a Suicide Gene for Treatment of Solid Tumors”, a novel gene which is important for the treatment of cancer. The project has involved many techniques in molecular and cellular biology and I have very successfully performed these experiments.

My research has yielded five outstanding scientific papers published or accepted for publication in highly ranked professional journals with international circulation such as Journal of Biological Chemistry, Molecular Pharmacology and Cancer Research etc. My publications contain not only original and scientific contributions but also practical and technical data and information useful for potential clinical use for cancer treatment. I gained the “doktorandtjanst” fellowship two times at Karolinska Institute because of my outstanding research achievements. Based on my important scientific discoveries, I defended my thesis three times, and got my Licentiate Degree (M.S) on February, 2001 [ Stockholm , Sweden ], First Ph.D. Degree on June, 2001 [Shenyang, P.R.China] and Second Ph.D. Degree on June, 2002 [Stockholm, Sweden], respectively.

Assistant professor, Department of Surgery, First Affiliated Hospital, China Medical University with Prof. Yulin Tian, I participated in 2 clinical research groups besides the general surgical oncology training. One was devoted to search for a more reliable technique to diagnose and treatment of the endocrine tumor of pancreas. Our group developed a systemic protocol to diagnose and treatment of endocrine tumor of pancreas basing our pathological and immunohistochemical study. Since this development, this protocol has been widely used clinically throughout China . For this, the Science and Technology Progress Prize for Clinical Achievement was given in 1996 by Shenyang Municipal Government, P.R.China. The other research group in which I was involved studied serious surgical infectious disease (SSID). The goal was to selecting appropriate and effective antibiotics to ensure low mobility and a high survival rate of serious surgical infectious disease. In recognition of our clinical efforts, First Place Award of Excellent Articles was given by Editorial Board of Chinese Journal of Practical Surgery in 1996.

In addition to my clinical duties, a significant amount of time has been devoted to teaching medical students and residents. Also, I assisted my mentors, Prof. Yulin Tian M.D. and Renxuan Guo, M.D./Ph.D., Both are very distingushed oncologists and act as editors of many of the top medical journals in China to critically judge the papers of others for publication.

M.S Graduate Student and Residency training in Department of Surgery, First Hospital, China Medical University, Shenyang, P. R. China.

Residency training in Department of Surgery, Liaoning Province Hospital , Shenyang , P. R. China.

In 1989, I got my M.D. from the Department of Medicine at the Dalian Medical University , P. R. China, with the best GPA among the 450 students in the department. Thereafter, I was admitted to a well-rounded, very competitive and demanding Surgical Oncology training program under the direction of Dr. Chengxun Liu, and Dr. Qi Dong in the Department of Surgery, Liaoning Province Hospital , which is the largest comprehensive cancer research and treatment center in the Northeast China . From here, I got very broad-based oncology training in clinical decision making, performance of complex operations, and development of advanced patient care skills.