Multiple Forms of Tyrosine Hydroxylase in Human Neuroblastoma Cells: Quantitation with Isoform-Specific Antibodies

Authors

  • John W. Haycock

    Corresponding author
    1. Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana, U.S.A.
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Address correspondence and reprint requests to Dr. J. W. Haycock, Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1100 Florida Avenue, New Orleans, LA 70119, U.S.A.

Abstract

Abstract: Human tyrosine hydroxylase (HTH) RNA undergoes alternative splicing, and four different forms of HTH mRNA have been previously identified. Rabbit antibodies were raised against octapeptides unique to each of the four isoforms of HTH predicted from these mRNAs. Blot immunolabeling of human adrenal medulla, pheochromocytoma, and several neuroblastoma cell lines with affinitypurified anti-HTH peptide antibodies demonstrated the presence of all four HTH isoforms in each of these tissues. Quantitative immunolabeling assays for HTH-1, -2, and -4 were established, and HTH isoform levels were determined in several human neuroblastoma cell lines. Whereas total HTH levels differed up to fourfold among the HTH-positive neuroblastoma cell lines studied [LA-N-1, LA-N-5, CHP-234, BE(2)-C, and BE(2)-M17], the relative abundances of HTH isoforms in each of the cell lines were similar. Immunocytochemical analyses demonstrated that HTH immunoreactivity was distributed unequally among the cells in each of these neuroblastoma lines, and morphological interconversion did not account for this heterogeneity. A direct relationship between the percentage of HTH-positive cells and overall HTH levels was also observed. This relationship, in the absence of an apparent clonal basis for the heterogeneity, suggests that HTH expression in neuroblastoma cells may be controlled in a relatively “all-ornone”(bimodal) fashion.

Abbreviations Used:
BTH

HTH, and RTH, bovine, human, and rat tyrosine hydroxylase, respectively

mRNAHTH

human tyrosine hydroxylase mRNA

MαRTH and RαRTH

mouse monoclonal and rabbit polyclonal antibodies to sodium dodecyl sulfate-denatured rat tyrosine hydroxylase

Ni/DAB

nickel-enhanced diaminobenzidine

PAGE

polyacrylamide gel electrophoresis

PBS

Dulbecco's phosphate-buffered, divalent-deficient saline

rHTH and rRTH

recombinant human and rat tyrosine hydroxylase, respectively

SDS

sodium dodecyl sulfate

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