SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Ryohei Hasegawa
First Department of Pathology,
Nagoya City University, Medical School,
Lecture, M.D. and Ph.D.

SPONSOR AND HOST INSTITUTION:
Dr. Samuel M Cohen
Department of Pathology and Microbiology,
University of Nebraska Medical Center
DATE OF VISITS: November 6 - 19, 1994

SUMMARY OF ACTIVITIES:
Carcinogen risk assessment has been part of one of the most ambitious public health programs ever carried out. Most of that activity in the U.S. has been centered in the U.S. Environmental Protection Agency (EPA). Well-operated long term carcinogencity tests have been conducted by the National Toxicology Program (NTP) as a part of risk evaluation. Since I have recently been involved In risk assessment based on my experience of carcinogenicity evaluation using our rat liver model, the purpose of my visit the U.S.A. was to learn the profiles of the agency and program and investigate possible issues in the carcinogenic risk assessment.
The topics I have selected before startling the visit were:
1. Issues on extrapolation from animal data to human.
2. Guidelines for carcinogencrty tests: present and future.
3. Classification of carcinogens based on carcinogenic mechanisms.
4. Combined effects of carcinogens how to evaluate.

1. Dr. S. M. Cohen, the University of Nebraska Medical Center, and Dr. J. Swenberg, University of North Carolina, have emphasized the importance of cell proliferation in the rat carcinogenesis by saccharin in the urinary bladder and!!!2u-globulln accumulation in the kidney. Although. Dr. Ward, NCI, Frederick, strongly warned that cell proliferation may be sufficient, but not always requisite, in carcinogenesis, the importance of cell proliferation may not to be reduced. Chemicals which induce increased cell profiration and carcinogenic in that organ must be carefully evaluated, especially in the cases of non-genotoxic chemicals.
   
2. The guidelines for carcinogenicity tests adopted by the U.S. Agencies are published from the NTP. The importance of long-term carcinogenicity studies for evaluation of carcinogenic potency will not be reduced even if they are very costly. However, replacement of the long-term test by economical ones such as medium-term bioassays we proposed is the issue which should be soon discussed as well as the establishment of methodology to evaluate carcinogenic mechanisms as supporting evidence. (Dr. Bucher, NIEHS)
   
3. Recently, International Federatin of Societies of Toxicologic Pathologists (IFSTP) proposed a classification of carcinogens. l= Carcinogens for man based on epidemiological data. 2= Genotoxic carcinogens for animals (strong or weak). 3= Epiogenetic carcinogens for animals (strong, weak, or not applicable in man). 4= Suspected carcinogens insufficiently tested. The similar classification of carcinogens based on carcinogenic mechanisms has also adopted by the IARC and then the NTP. Although the terminology must be carefully selected; DNA-reactive and DNA non-reaviive carcinogens are more recommendable, it is update common sense to distinguish these two types of carcinogens since the risk evaluation is clearly different between them. It is not always possible to evaluate or prove carcinogenic mechanisms in each case. Beside the long-term tests, additional assays for evaluate carcinogenic mechanisms are now important but it is not always required by the agencies. (Dr. Farland, EPA; Dr. Ward, NCI)
   
4. I met with Dr. C. Brown, a statistician of NCI, Rockville, and discussed appropriate analysis for chemical mixtures and the special terms of synergism, such as autosynergism, heteroadditive and isoadditive. Our mathematical methods were confirmed its rightness.
   

I am very thankful th the US-JAPAN Program for giving me the unique opportunity to meet with researchers and officials of this field in the U.S.A. This experience will be very beneficial for me to consider the risk assessment in Japan.

Seminars:
Nov. 10. At NIEHS “Medium term liver bioassy for carcinogenic agents and issues of heterocyclic amines”
Nov. 14. At NCI, Frederick. “The carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), a mutagenic chemical of cooked food orgin in rats: Medium-term liver bioassay and a tranplacental study”
Nov. 16. At NCI, Bethesda. “PhIP carcinogenicity in rats: Medium-term liver bioassay and transplacental carcinogenicity”



(2) Narito Morii
Department of Pharmacology,
Kyoto University Faculty of Medicine
Associate Professor, M.D. and Ph.D.

SPONSOR AND HOST INSTITUTION:
Dr. Raymond B. Birge,
Department of molecular Oncology,
The Rockefeller University
DATES OF VISIT: November 6 - 18, 1994

SUMMARY OF ACTIVITIES:
Role of the rho gene product in tumor cell invasion: rho p21 and protein tyrosine phosphorylation signaling.
Cells undergo adhesion to extracellular matrix or other cells in response to external stimuli. We have analyzed signaling mechanism of this process and revealed that a small GTP-binding protein, rho p21 lies in this signaling. rho p21 is a member of the ras superfamily of small GTPases. The function of this protein have been examined by the use of botulinum C3 exoenzyme, which specifically ADP-ribosylates and inactivities this protein. Studies using this exoenzyme revealed that rho p21 mediates cellular processes of stimulus-envoked cell adhesion in fibroblasts, platelets and lymphocytes, and cell motility in leukocytes and fibroblasts. Cell adhesion plays a fundamental role in pathological processes such as tumor invasion and metastasis. Recently some of oncogenes have been identified as putative GDP/GTP exchange factors for rho p21 and/or its subfamily proteins. This finding suggests that rho p21 plays a role in tumor promoting and invasion. We have previously demonstrated that rho p21 in involved in growth factor-induced protein tyrosine phosphorylation. In this study we examined signaling pathway of rho p21 and its functional role in protein tyrosine-phosphorylation signaling.
[Enperimental Procedures and Results]

1. Interaction of rho p21 with Crk adaptor protein: Effect of ADP-ribosylation of rho p21 on cell shape was studied in v-crk transformed PC12 cells which was established by Dr. Birge of the Rockefeller University (Mol. Cell. Biol. 14, 1964; 1994). The v-crk-PC12 undergoes neurite growth in response to EGF as well as NGF. C3 treatment did not affect this aberrant induction of neurite growth by EGF or cell shape in unstimulated conditions. We previously revealed that inactivation of rho p21 causes neurite outgrowth in PC21 cells. This finding suggests the possibility that inactivation of rho p21 is accelerated in v-crk transformed cells. To assess this, GAP activities for rho p21 in crude cell homogenate and v-Crk precipitates were examined. No significant difference, however, was found between v-crk transformed and untransformed control cells. These results suggest that v-Crk in the transformed cells dose not interact with rho p21 signaling.
   
2. Role of rho p21 in bombesin- and endothelin-induced tyrosine phosphorylation in cultured fibroblasts: We previously found that rho p21 is involved in protein tyrosine phosphorylation in response to lysophosphatidate (LPA) in Swiss 3T3. This finding was examined in bombesin- and endothelin-stimulated fibrablasts. Both mitogenic peptides induced the tyrosine phosphorylation of pl 25 FAK and p72 paxillin. This phosphorylation was significantly attenuated by the treatment of cells with C3 enzyme before challenge of the cells to the agonists. This result suggests the presence of rho-dependent activation of some tyrosine kanases.
   
3. Renaturation assay to detect the rho p21-dependent kinase(s): We renatured kinase activity on protein blots and analyzed protein kinase activation by LPA in cultured mouse Swiss 3T3 and rat 3Y1 fibroblasts. LPA induced the activation of several kinases with apparent molecular weights of 145K, 85K, 64-65K (a doublet), 60K and 43K in a time-dependent manner (tentatively named p145, p85, p64/65, p60 and p43 kinase, respectively). The p43 kinase was Identified as a mitogen activated protein(MAP)-kinase. Phosphoamino acid analysis revealed that all of these kinases belonged to the family of the protein-serine/threonine kinases. Treatment of the cells with pertussis toxin significantly attenuated the LPA-induced activation of MAP-kinase, whereas botulinum C3 exoenzyme attenuated the activation of p145, p85, and p64/65 and MAP-kinases. These results suggest that LPA activated p145, p88 and p64/65 kinases by a pertussis toxin-insensitive rhop21-dependent pathway and that the activation of MAP-kinase was mediated by both the pertussis toxin-sensitive and the pertussis toxin-insensitive rho p21-dependent pathways.