Dominant erythrocytosis and EPO hypersensitivity. (A) Pedigree of the Swedish family. (B) EPO-dependent proliferation in 32D cell lines. Cells expressing WT (•), mFin (▴), mSwed (▪), mFin+WT (▾), or mSwed+WT (⧫) were assayed for growth in EPO-containing medium. (C and D) EPO-dependent Jak2 and Stat5 activation in 32D cell lines. Cells were stimulated with EPO and Jak2 and Stat5 activation determined by immunoprecipitation and antiphosphotyrosine immunoblot assays. Methods: The inheritance pattern of familial erythrocytosis among members of a 5-generation family from southern Sweden was determined by clinical observation. In some cases the presence of the mutant EPOR allele was assayed by direct genomic sequencing or an allele-specific hybridization test. 32D cell lines were established as described.12 Cells were grown in media containing serial dilutions of EPO (from 10−4 to 10 U/mL), or in media containing interleukin-3 (IL-3). Viable cells were counted after 3 days. The number of cells in EPO cultures is expressed as a percentage of the cells present in IL-3 cultures (% IL-3 response). For Jak2 and Stat5 activity assays, 32D cells were stimulated for 10 minutes at 37°C with 0.2, 1.0, 2.0, or 10.0 U/mL EPO, or were left unstimulated, as indicated. Proteins were immunoprecipitated from detergent cell extracts with an antibody specific for Jak2 or Stat5, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The filters were probed with antiphosphotyrosine antibody 4G10 and developed with chemiluminescent reagents.9 12 A computer-generated image of the region of the gel containing Jak2 (≈130 kD) or Stat5 (≈90 kD) is shown. Chemiluminescent signals were quantified on a lumi-imager and are expressed as a percentage of the level of Jak2 or Stat5 phosphorylation elicited by EPO stimulation of the WT receptor at 10 U/mL EPO (arbitrarily set to 100%). The levels of protein phosphorylation were normalized to the levels of immunoprecipitated Jak2 or Stat5 for each sample.
