(1) Shun Nakamura
The Institute of Medical Science
The University of Tokyo, Tokyo
Sponsor and Host Institution:
Dr. G. Vande Woude Litton-Bionetics, Inc. NCI-Frederick Cancer Research Facility Frederick
Dr. E. Gilboa Princeton University Princeton
Dr. K. Arai DNAX Research Institute
Dates of Visit: July 9- October 9, 1985
Summary of Activities:
1. July 9, 1985- July 13, 1985: I attended the first annual meeting on oncogenes at Frederick.
2. July 14- August 11: I visited Dr. G. Vande Woude's laboratory to learn the handling of the retrovirus vector system. Both of us were interested in the correlation between the level of the expression of c-Haras gene and the extent of transformation of the cells. I inserted a human c-Haras cDNA into the cloning site of the retrovirus vector developed by Dr. Vande Woude's laboratory. The expression of the ras gene was designed to be controlled by mouse mammary tumor virus promoter. The construction of the recombinant virus was completed. However, the isolation of cells expressing the ras gene has not been completed. I will continue this work in Japan and keep in contact with Dr. Vande Woude.
3. August 12- September 7: I visited Dr. E. Gilboa's laboratory to work on the suicide retrovirus vector system developed by Dr. Gilboa. He made a suicide vector which loses 5' LTR function after infection because of the deletion of a part of the U3 structure of 3' LTR. This vector would be ideal for gene therapy use because there is less chance of activation of c-oncogenes by LTR insertion. To develop the vector for gene therapy, as well as for the general tool for gene transfer into animal cells, both of us were interested in determining the condition which minimizes interference between two promoters on the vector and maximizes the expression of the inserted gene. Two promoters, one for the expression of a selectable gene and the other for the inserted gene, interfere with each other and the interference may reduce the level of expression of the inserted gene or may yield the deletion of the promoter of the inserted gene. Therefore, I started to construct a series of the recombinant virus to further examine this condition. I will continue the work and collaboration with Dr. Gilboa.
4. September 8- October 9: I visited Dr. K. Arai's laboratory to learn the handling of cDNA expression vector system developed by Dr. H. Okayama and P. Berg. Dr. Arai's laboratory has successfully isolated a couple of lymphokine genes by screening lymphokine activity expressed in animal cells which had been transfected with cDNA library. I plan to use the cDNA expression vector to isolate the genes which amplify the growth promoting signal transduced by the ras gene product as well as the genes which suppress the ras gene function in primary cultured cells or neuron cells.
(2) Masuo Yutsudo
Department of Tumor Virology Research Institute for Microbial Diseases Osaka University, Osaka
Sponsor and Host Institution:
Dr. Peter M.Howley Laboratory of Tumor Virus Riology National Cancer Institute
Dates of Visit: November 2- December 1, 1 985
Summary of Activities:
(I) Studies
We isolated and molecularly cloned various types of human papillomavirus (HPV) from patients with epidermodysplasia verruciformis (EV). Further, we showed that some of these HPVs (HPV-17 and 20) were involved in skin carcinogenesis. However, because of the lack of an in vitro system for replication of or transformation by HPV, there was little progress on the mechanism of carcinogenesis by HPV. The objectives of study at, Dr. Howley's laboratory were I ) to learn the procedures for transformation of mouse cell line C127 by bovine papillomavirus (BPV) DNA, and 2) to test the transforming activity of HPV DNA which we cloned from EV patients using C127 cells. I accomplished the first objective. C127 cells, which were plated the previous day at 2-3x105 cells/60mm dish, were fed with fresh medium 3 hrs. before addition of DNA-Ca precipitate. DNA (1µg of BPV DNA and 19µg of salmon sperm DNA) was mixed with CaCl2 in Hepes-buffered saline, allowed to stand at room temperature for 30 min., and added into the plate (0.5ml). Four hours later, the cells were treated with 15% glycerol for 1 min. and were incubated overnight in medium containing 5mM sodium butyrate. The medium was changed 2-3 times per week. At 10 days after transfection, about 30-50 foci per plate appeared. We were satisfied with this transformation efficiency. Regarding objective 2, we could not observe any transformed foci of C127 cells until 2 weeks after transfection with HPV DNAs. I used DNAs of four types of HPVs: HPV-3, 14, 17 and 40. All these HPVs were isolated from patients with EV, and HPV-14 and 17 were shown to have a close relation to skin carcinogenesis. However, these HPV DNAs did not transform C1 27 cells under the same conditions of transfection as those used for BPV DNA. In the future, we will try different experimental conditions to evaluate the trans-forming activity of HPV DNA; for example, transfection must be performed in the presence of cofactors, or cells must be cotransfected with HPV DNA and the neo gene and followed by injection into nude mice, etc.
(II) Information
l. Laboratory of Dr. Howley (NCI,Bethesda)
This laboratory has introduced mutations in vitro into each gene of BPV-l and showed that the E5 gene had transforming ability. Further, they detected 7.5Kd presumptive E5 protein using antiserum against the synthetic peptide of the N-terminal portion of the E5 open reading frame.
This laboratory has found that when the E2 gene of BPV-1 coexisted with the CAT gene which was ligated downstream of regulatory region of BPV-1, CAT activity increased, suggesting transactivation ability of the E2 gene of BPV-1.
2. Laboratory of Dr. Lowy (NCI, Bethesda) This laboratory has shown that the E6 gene of BPV-1 recombined with the LTR of retrovirus transformed C127 cells. They next prepared antiserum against the fusion protein between CII of!!
!phage and E6 and detected 16-17Kd of the presumptive E6 protein. 3. Laboratory of Dr. Lancaster (Georgetown University, Washington, DO This laboratory has isolated new types of HPVs from tissues of cervical mild dysplasia (HPV-3 1 and 35).
4. Laboratory of Dr. Shah and Dr. Sawada (Johns Hopkins University, Baltimore) The Investigators in this laboratory have studied the epidemiology of various types of condylomata and collected basic data on the morphology of condylomata and the mode of their transmission.
5. Laboratory of Dr. Maniatis (Harvard University, Cambridge) This laboratory has developed the method for detecting a single base change in DNA based on the fact that the heteroduplex and homoduplex of the DNA fragment could be separated by electrophoresis.
I would like to utilize this method to identify any small change in HPV DNA existed as oligomeric plasmid in skin carcinoma tissues.
(3)Tada-aki Hori
Division of Genetics
National Institute of Radiological Sciences, Chiba
Sponsor and Host Institution:
Dr. Thomas W. Glover University of Michigan Ann Arbor
Dates of Visit: November 25- December 25, 1985
Summary of Activities.
Interest has recently grown in the possible role of chromosomal fragile sites (FS) as factors predisposing to chromosome rearrangements characteristic of specific human cancers. A heritable FS on human-X chromosome is known to be associated with mental retardation, called fragile X syndrome. In collaboration with Dr. Glover, we have constructed somatic cell hybrids between fragile X cells and thymidylate synthase-negative mouse mutant cells. In thymidine-auxotrophic hybrid cells, we have shown that thymidine de-privation alone was effective in inducing the expression of FS and also suggested that the mutation in FS itself is responsible for the FS expression. The objective of my visit was to exchange and discuss recent information on heritable FS. I visited the following three institutions and presented seminars on our recent results mentioned above.
I ) Dr. White's group at the University of Utah Medical School
The main purpose of this visit was to obtain new information on the fragile X syndrome. The most current problem is its genetic heterogeneity. I have learned there exists at least two kinds of families which differ in terms of the rate of recombination between FS and F9 which is closely linked to FS. We agree we need to compare the mode of expression of FS derived from genetically distinct families in our system and to analyze the nature of mutations by applying RFLPs of closely linked DNA markers which are available in his group. I also had a chance to discuss, with Dr. Lark, genetic assay systems of recombination in mammalian somatic cells.
II) Dr. Latt's group at Harvard University Children's Hospital
In this laboratory, we discussed the strategy for cloning the fragile X site and obtained valuable information. As a result, we planned a collaborative study on the isolation of the fragile X chromosome from our human-mouse hybrid cells which retain the fragile X chromosome as the only ' human chromosome, by using his method of chromosome sorting. I also had a chance to talk with Dr. Little about the chromosome instability of cancer-prone human genetic disorders and radiation-induced chromosome abberrations.
III) Dr. Glover's group at the University of Michigan Medical Center
The purpose of this main visit was to exchange new information on the possible involvement of FS in cancer-specific chromosomal rearrangements and to discuss future plans of our collaborative study. I have learned that in the case of acute nonlymphocytic leukemia, 8 out of 1 6 patients with inv (16) (p13q22) were found to be carriers of FS at 16q22, including our own data. This appeared to be very significant in cancer research. During this stay, we did short-term experiments to see the timing of DNA replication of FS on the X chromosome in our thymidine-auxotrophic hybrid cells. Thymidine starvation was effective in cell synchronization. The FS was found to be replicated late in the S phase. With these results, we planned a collaborative study on the cloning of FS. I also had an opportunity to learn, from Dr. Collins, a new technique of "chromosome hopping" which seems to be very useful for the isolation of heritable FS. I am very grateful to the U.S.-Japan Cooperative Cancer Research Program for their full support of my fruitful visit to the U.S. which stimulated our research greatly and enabled me to initiate new collaborative projects with the above-mentioned research groups.
(4) Yuji Kurokawa
National Institute of Hygienic Sciences
Tokyo
Sponsor and Host Institution:
Dr. Thomas J. Slaga The University of Texas System Cancer Center Smithville, Texas
Date of Visit: December 8- December 19, 1985
Summary of Activities:
There is now accumulating evidence that suggests the involvement of active oxygen radicals in the mechanism of carcinogenesis. I have found that potassium bromate is carcinogenic and induces renal cell tumors in rats. Potassium bromate is used as a food additive utilizing its oxidizing property, and the involvement of active oxygen species may be involved in its carcinogenesis. Therefore, the objectives of this visit were: firstly, to discuss the problems regarding the carcinogenic and promoting activities of oxidizing chemicals from both the pathological and the biochemical points of view; secondly, to present my own work on the carcinogenic and promoting effects of potassium bromate as a seminar. A summary of the discussion with the researchers follows.
Dr. J. Reddy (Department of Pathology, Northwestern University): His studies deal mainly with the induction of hepatomas in animals by treatment with hypolipidemic drugs. He found extensive proliferation of peroxisomes in liver cells and subsequently suggested that hepatomas were induced by hydrogen peroxide generated from the peroxisomes. Our discussion focused on the carcinogenic and promoting action of hydrogen peroxide in vivo. We agreed that more experiments will be necessary to elucidate the action of hydrogen peroxide in carcinogenesis.
Dr. W. Troll (New York University Medical Center): He has been involved, for many years, in studies on the mechanism of promotion, especially that shown by TPA. He now has a theory that TPA induces a respiratory burst in polymorphonuclear leukocytes associated with the production of various oxygen radicals that results in promotion of skin carcinogenesis. Following my seminar, we discussed the need for animal experiments to facilitate the mechanism of carcinogenic and promoting effects exhibited by oxidant chemicals.
Dr. H. Demopoulos (New York Ukiversity Medical Center): He stated the importance of antioxidants as a major factor for the inhibition of carcinogenesis based on his wide research on free radical pathology. Indeed, he started the so-called "high-dosage antioxidant therapy" for volunteers for prophylaxis of various diseases, including cancer.
Dr. T. J. Slaga (University of Texas System Cancer Center): His work has concentrated on two-stage skin carcinogenesis using Sencar mice that he developed. I was interested to learn that he recently found that benzoyl peroxide, known as an oxidizer for multiple purposes, was effective as a second stage promoter of mezerein, which he also had identified earlier. Therefore, I believe it will be useful to analyze the role of oxidizing chemicals in the stages connecting promotion and progression, i.e., malignant transformation from benign to malignant state. Also, he suggested that the measurement of chemiluminescence in vitro will be a useful technique for comparing the biological activity of many oxidants.
Specific Comments:
1. I reconfirmed that many more animal experiments will be needed to clarify the mechanism underlying the carcinogenic and promoting effects shown by the oxidant chemicals.
2. I believe that it is really important to continue the collaborative work on the biochemical and morphopathological studies to analyze the effect of active oxygen species in carcinogenesis.
3. I realize that, through my seminar, the researchers could have information on the present status of the cooperative work on animal carcinogenicity testing of environ-mental chemicals in Japan under the subsidy of the Ministry of Health and Welfare of Japan.
(5) Kazuo Negishi
Okayama University
Okayama
Sponsor and Host Institution:
Dr. Robert D. Wells The University of Alabama at Birmingham Birmingham
Dates of Visit: January I O- March 9, 1 986
Summary of Activities:
I worked on "Z-form DNA and Cancer" in the Department of Biochemistry at the University of Alabama at Birmingham during the above period. I also visited Dr. U. Raj-Bhandary of the Massachusetts Institute of Technology for two days and discussed this subject with him.
Alternating purine and pyrimidine sequences in DNA can generate a Z-form of the DNA under specified conditions. The Z-DNA can be attacked by some direct carcinogens more easily than normal right-handed B-DNA. It has been suggested that the conformation of the junctions between B-DNA and Z-DNA (B-Z junctions) is different from normal double strand DNA because B-Z junctions are sensitive to nuclease S1 which cuts single strand regions of nucleic acids. As the first experiment, I studied the chemical reaction with two reagents, bisulfite-hydrazine mixture and permanganate which are specific for single strand nucleic acids, toward Z-DNA induced in plasmid DNA. The plasmids used had been constructed by R. D. Wells and co-workers.
The plasmids pRW756 and pRW777 contain the (dG-dC)16 and (dT-dG).(dC-dA)32 sequences in their DNA, respectively. These sequences undergo a supercoil-induced transition from a right- to left-handed helix. The supercoiled pRW756 DNA reacted with the bisulfite-hydrazine mixture and the DNA was further treated with ethylacetimidate to fix the modification. After the linearization by restriction endonuclease, PstI or Aval, the DNA was digested by nuclease Sl to localize the modification sites. Electrophoretic analysis revealed that the bisulfite-hydrazine modified nucleotides in the B-Z junction(s) and those in a cruciform in a specific manner. It was also shown that potassium permanganate reacted with nucleotides at the B-Z junction of pRW777 DNA. These lines of evidence strongly indicate that B-Z junctions have unpaired bases. Some additional work is required to obtain conclusive results.
Dr. Wells and I have been encouraged by these observations, and we have decided to continue this project. Therefore, I am now doing experiments to determine the optimal conditions for these reactions, while Dr. Wells is making efforts to find effects of this modification on the superhelicity of the plasmid DNA.
Specific Comments
1. We studied chemical modification of nucleic acid and are interested in the conformational changes of DNA by B-Z transition. The program enabled me to work in a laboratory in which Z-DNA has been studied extensively. If not, it would be difficult for us to obtain and to characterize DNA containing Z-DNA.
2. I found that the reagents which were developed by our group in Japan are useful as probes for B-Z junctions. Therefore, we will continue our collaboration with Dr. R. D. Wells.
3. We showed that bisulfite-hydrazine mixture and potassium permanganate can modify B-Z junctions specifically. This observation indicated the possibility that B-Z junctions might be a specific target for some DNA damaging agents and that the modification might play some role in the initiation of carcinogenesis.
(6) Koichi Yoshida
Department of Molecular Biology Cancer Research Institute
Sapporo Medical College, Sapporo
Sponsor and Host Institution:
Dr. G.Chinnadurai Institute for Molecular Virology St. Louis University Medical Center St. Louis
Dates of Visit: March 8, 1985- March 7, 1986
Summary of Activities:
Transformation of rodent cells by adenovirus requires expression of the viral early region E1A and E1B transcription units. In particular, the function(s) encoded by E1A is required for the establishment of cells growing indefinitely in vitro. In addition, E1A can collaborate with the E1B gene to transform cultured primary cells completely. Here, I made plasmid DNA designed to probe the function of two major E1B proteins, the 19 K and the 55 K. To determine which E1B proteins collaborate with E1A gene products in cell transformation, adenovirus 7 (Ad 7) ElB plasmid with specific mutations in the genes coding for the two proteins were constructed. Upon transfection of primary baby rat kidney cells (BRK) with an Ad 7 or Ad 2 E1A plasmid and a plasmid carrying the normal 19 K protein but mutated 55 K protein, transformation efficiency increased with four-to five-fold of EIA plasmid alone. In contrast, a plasmid carrying the normal 55 K protein but the mutated 19 K protein transformed BRK cells with only 1/2 to 1/3 of the efficiency of EIA plasmid alone. Since E1B plasmid does not show any transforming activity, the result indicates that the 19 K protein of the E1B gene is required for efficient transformation in collaboration with products of the EIA gene. One of the functions of the EIA gene product is a negative regulator of some viral and cellular enhancers, including polyoma and SV40 enhancers. Using a transient expression assay of CAT gene in HeLa cells and Ad 2 E1A-transformed BRK cells, I showed that repression of transcription from the SV40 enhancer by the EIA gene products can be released by the Ad 2 and Ad 7 19 K protein, one of the two major E1B proteins. Hence, the 19 K protein is assumed to be an activator of viral and cellular genes which are regulated negatively by E1A gene products. One approach to the elucidation of the modes of action of Ad E1A and ElB gene-encoded proteins in cell transformation involves the use of highly transmissible murine leukemia virus vector. This enabled us to transfer individual Ad transforming gene products into cell in vitro and into the whole animal. Here, I made plasmids of defective murine leukemia virus proviral DNA (pMSVgpt), which are carrying cDNA copy of 12s or 13s E1A mRNA of Ad 2. Transfection of these plasmids into an MLV-packaging cell line (!!
!2) and isolation of recombinant retroviruses are now in progress. Specific Comments:
1. I could get useful plasmids and cell lines from the laboratories I visited as well as other laboratories. I had a chance to see the laboratories and research facilities of Monsanto Company in St. Louis and attended several research and specialized seminars.
2. When problems occurred, I received good advice and suggestions from the professor and colleagues. I will work with the same lab for another year to complete initial achievement of this program. It seems to be possible to collaborate with the Institute for Molecular Virology of St. Louis University in the future.