Member, Cell Stress Biology
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo NY 14263
Telephone: (716) 845-3147
Fax: (716) 845-8899
E-mail: john.subjeck@roswellpark.org

Professor
Molecular & Cellular Biophysics and Biochemistry
Roswell Park Graduate Division
University at Buffalo

Dr. John R. Subjeck joined the staff of Roswell Park Cancer Institute (RPCI) in 1979, as a Cancer Research Scientist, Division of Radiation Biology, Department of Radiation Medicine. He was appointed Member in the departments of Cell Stress Biology and Molecular and Cellular Biology in 1999, and is also a Professor of the Molecular & Cellular Biophysics and Biochemistry Roswell Park Graduate Division, University at Buffalo (UB).

Dr. Subjeck earned his doctoral degree in Biophysical Sciences, UB School of Medicine & Biomedical Sciences in 1974. He completed a postdoctoral fellowship in Experimental Pathology at RPCI in 1977. From 1977-1979, Dr. Subjeck specialized in Hyperthermia as a Research Affiliate in Medical Physics in the Division of Radiation Biology at RPCI. He is certified by the American Board of Radiology in Therapeutic Radiological Physics.

Dr. Subjeck’s research interests focus on heat shock/stress proteins

Projects
Heat Shock Proteins and Hyperthermia

Stress proteins are known to play essential roles in normal cellular functions and are involved in numerous pathways involving protein processing and interactions. They correspondingly protect cells from a variety of protein and cellular damaging environments at the molecular, cellular and organismal levels. Several of the stress proteins have come under intense scrutiny.

However, from the earliest studies, major stress proteins known as hsp110 (in the cytoplasm) and grp170 (in the endoplasmic reticulum) were observed, but studies of their structure and function were not undertaken; that is, until recently. Cloning of these proteins in this laboratory indicated that they are related to one another and are also "distant" relatives of the intensively studied hsp70 family and gave been observed in every eucaryotic cell examined, yeast to man. It is our overall goal to determine the functions of these ubiquitous stress proteins and how they compare to and interact with the hsp70s as well as other molecular chaperones. Secondly, we are studying hsp110 and grp170 as potential cancer vaccines.

We have demonstrated that both hsp110 and grp170 are highly efficient peptide chain binding proteins and form complexes with other full length proteins under heat shock conditions. It has been shown that some stress proteins possess have specific roles in the immune response. We are developing a novel vaccine approach for the use of hsp110 and grp170 in cancer therapy. This specifically utilizes synthetic vaccines composed of recombinant hsp110/grp170 and tumor associated proteins. Moreover, we are investigating the molecular/cellular mechanisms by which hsp110, hsc70 and grp170 interact with antigen presenting cells in eliciting an anti-tumor immune response.

Progress
Heat shock proteins are often observed to bind to denatured protein and inhibit protein aggregation. The ability of recombinant, full-length hsp110 to perform this function has been previously assessed using two reporter proteins, luciferase and citrate synthase. It was found that hsp110 inhibits heat induced protein aggregation and holds denatured protein in a folding competent state in a manner similar to hsc70, but is far more efficient than hsc70 in carrying out this important chaperoning function.
We have subsequently identified the functional domains of hsp110 which are responsible for its chaperoning activity by targeted deletion mutagenesis, using the known structure of DnaK as the model. The chaperoning ability of mutants was again assessed based on their ability to solubilize heat denatured luciferase as well as to refold luciferase in the presence of rabbit reticulocyte lysate. It was shown that these functions require only an internal region of hsp110 which includes the predicted peptide binding domain and two immediately adjacent C-terminal domains.
It is also shown that hsp110 is approximately four fold more efficient in stabilizing demature protein than is hsp70. Similar studies of the structure and function of grp170 have also been completed. Grp170 exhibits many of the structural/functional properties of hsp110, while also differing in important ways. Grp170 is also a potent peptide chain binding protein.
Lastly, several studies have confirmed that certain stress proteins can function as potent vaccines against a specific cancer when purified from the same tumor. This is based on the peptide binding characteristics of some stress proteins and their apparent involvement in antigen processing pathways and clinical trials using this approach are underway at other centers.

Likewise, we have also shown that vaccination with hsp110 or grp170 purified from Meth A fibrosarcoma caused complete regression of this tumor in mice. Since clinical use of "heat shock vaccine therapy" can be limited by the quantity of hsp/grp which can be purified from a human tumor (surgical) specimen, we have begun the characterization of synthetic, recombinant protein based vaccines. These studies are founded in our understanding of the structural/molecular chaperoning properties of hsp110 and grp170, referred to above, and the mechanisms of interaction of hsp110/grp170 with cells of the immune system. Recent data indicate that recombinant, molecular-targeted vaccines elicit powerful and specific anti-tumor CTL responses, suggesting that this may represent a new and highly potent approach to cancer therapy.

Key Publications

• Subjeck, J.R. and Sciandra, J. Co-expression of heat shock proteins and thermotolerance in mammalian cells. In: Heat Shock, From Bacteria to Man. Cold Spring Harbor Monograph Series, Tessieres, Ashburner and Schlessinger (eds.), pp. 405-411, 1982.
  
• Sciandra, J., Hughes, C. and Subjeck, J. Induction of glucose regulated proteins during anaerobic exposure and heat shock proteins during reoxygenation. Proc. Natl. Acad. Sci. USA 81: 4843-4848, 1984.
  
• Yoon, Y., Easton, D., Murawski, M., Chen, X., Burd, R. and Subjeck, J. Identification of a new family of HSP70 like proteins through the cloning of the 110 kDa heat shock protein. J. Biol. Chem. 270:15725-15733, 1995.
  
• Chen, X., Easton, D., Oh, H.-J., Lee Yoon, D.S., Liu, X. and Subjeck, J. The 170 kDa glucose regulated stress protein is a large HSP70-, HSP110-like protein of the endoplasmic reticulum. FEBS Lett. 380:68-72, 1996.
  
• Oh, H.J., Chen, X. and Subjeck, J. Hsp110 protects heat denatured proteins and confers cellular thermoresistance. J. Biol. Chem. 272:31636-31640, 1997.
  
• Di, Y.P., Repasky, E. and Subjeck, J. The distribution of hsp70, protein kinase C and spectrin is altered in lymphocytes during a fever-like hyperthermia exposure. J. Cell. Physiol. 172:44-54, 1997.
  
• Wang, X., Kazim, L., Repasky, E. and Subjeck, J. Cancer vaccine activity of hsp110 and grp170 and enhancement of efficiency by fever like hyperthermia. J. Immunol. 165: 490-497, 2001.
  
• Manjili, M.H., Li, Y., Wang, X.Y., Henderson, R., Repasky, E. and Subjeck, J.R. Development of a recombinanthsp110-Her 2/nue cancer vaccine using the natural chaperoning properties of hsp110. Cancer Res. 62:1737-1742, 2002.
  
• Wang XY, Chen X, Manjili MH, Repasky E, Henderson R, Subjeck J. Targeted immunotherapy using reconstituted chaperone complexes of heat shock protein 110 and melanoma-associated antigen gp100. Cancer Res., 15;63(10): 2553-60, 2003.
  
• Manjili, M.H., Wang, X-Y., Chen, X., Martin, T., Repasky, E., Henderson, R. and Subjeck, J. HSP110- HER-2/neu chaperone complex vaccine induces protective immunity against spontaneous mammary tumors in HER-2/neu transgenic mice. J. Immunol. 171(8):4054-4061, 2003.
  
• Park, J., Easton, D., MacDonald, I. Chen, X., Wang, X., and Subjeck, J. The chaperoning properties of mouse grp170, a member of the third family of hsp70 related proteins. Biochemistry, 42(50):14893-14902, 2003.
  
• Manjili, M.H., Wang, X.Y., MacDonald, I.J., Arnouk, H., Yang, G.Y., Pritchard, M.T., Subjeck, J.R. Cancer immunotherapy and heat-shock proteins: promises and challenges. Expert Opin Biol Ther., 4(3):363-73, 2004.