We have established that the L-system of amino acid transport is invariably, and markedly impaired in the lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL). This highly specific difference between CLL B-cells and normal B-cells will be exploited in an effort to isolate and characterize the protein or proteins responsible for l-system transport. The membrane proteins from normal and CLL B-lymphocytes will be separated using two dimensional gel electrophoresis. Two methods will be used to identify the L-system transport protein or proteins on two dimensional gels. Monoclonal antibodies that react specifically with the L-system will be generated by making mice tolerant to CLL B-lymphocytes which are deficient in the L-system and subsequently immunizing the mice with normal B-lymphocytes which have an intact L-system. The monoclonal antibodies will be screened for reactivity to normal but not CLL B-cells. Monoclonal antibodies binding to normal B but not CLL B-cells will be screened for functional inhibition of the L-system. Such antibodies can be used to extend the sensitivity of two dimensional gel electrophoresis for localization of the L-system transport protein. In addition, photoreactive radiolabelled amino acids which can be bound specifically to the L-system site will be used to identify the transport protein on two dimensional gels. As an extension of previous investigations we will determine whether the L-system transport in the T-lymphocyte population in CLL-B-cell leukemia accounts for the 10% residual transport that is measured. We will separate the T-lymphocyte population from B-cell CLL blood using a sepharose anti-immunoglobulin affinity column, and the measurements of L-system transport will be correlated with the cytogenetics of the B and T-cell populations. Further, CLL B-cells will be treated with mitogenic (cytochalasin B, staph protein A) and maturational (phorbol esters) agents to determine the effect of 1) new DNA synthesis or 2) expression of the plasma cell phenotype on the ability to acquire a heightened capacity of L-system transport. These studies will assess whether the impaired L-system is related to a diminished proliferative capacity or arrested maturation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA034691-06
Application #
3172460
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1983-06-01
Project End
1989-06-30
Budget Start
1988-06-01
Budget End
1989-06-30
Support Year
6
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Rochester
Department
Type
School of Medicine & Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
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Woodlock, T J; Young, D A; Boal, T R et al. (1993) Phorbol ester-induced membrane proteins in chronic leukemic B-lymphocytes. Candidate proteins for the L-system amino acid transporter. J Biol Chem 268:16020-7
Segel, G B; Boal, T R; Cardillo, T S et al. (1992) Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA. Proc Natl Acad Sci U S A 89:6060-4
Woodlock, T J; Brown, R; Mani, M et al. (1990) Decreased L system amino acid transport and decreased gamma-glutamyl transpeptidase are independent processes in human chronic lymphocytic leukemia B-lymphocytes. J Cell Physiol 145:217-21
Liang, S L; Woodlock, T J; Whitin, J C et al. (1990) Signal transduction in N-formyl-methionyl-leucyl-phenylalanine and concanavalin A stimulated human neutrophils: superoxide production without a rise in intracellular free calcium. J Cell Physiol 145:295-302
Woodlock, T J; Segel, G B; Lichtman, M A (1989) Diacylglycerol and calcium induce rapid enhancement of A-system amino acid transport by independent mechanisms in human T-lymphocytes. J Cell Physiol 141:33-9
Segel, G B; Woodlock, T J; Murant, F G et al. (1989) Photoinhibition of 2-amino-2-carboxybicyclo[2,2,1]heptane transport by O-diazoacetyl-L-serine. An initial step in identifying the L-system amino acid transporter. J Biol Chem 264:16399-402
Costa-Casnellie, M R; Cragoe Jr, E J; Segel, G B et al. (1989) Activation of the Na+/H+ exchanger by phorbol ester and osmotic shock is dependent on the degree of neutrophilic maturation. J Cell Physiol 141:294-300
Costa-Casnellie, M R; Segel, G B; Lichtman, M A (1988) The Na+/H+ exchanger in immature and mature granulocytic HL-60 cells. Property changes induced by intracellular acidification and cell maturation. J Biol Chem 263:11851-5
Simon, W; Segel, G B; Lichtman, M A (1988) Upper and lower time limits in the decision to recommend marrow transplantation for patients with chronic myelogenous leukaemia. Br J Haematol 70:31-6

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