Steven C. Clark

Molecular characterization and expression of the gene encoding human erythroid-potentiating activity.

Erythropoietin is the primary physiological regulator of erythropoiesis; however, in vitro studies have identified another class of mediators which appear to be important in stimulating erythroid progenitors. These factors have generally been referred to as burst-promoting activities (BPA), because they stimulate the growth of early erythroid progenitors referred to as burst-forming units-erythroid (BFU-E) which give rise to colonies of up to thousands of haemoglobinized cells1,2. We recently reported purification of a burst-promoting activity from medium conditioned by the Mo T-lymphoblast cell line infected with human T-cell lym-photropic virus type II (HTLV-II)3,4. This purified glycoprotein of relative molecular mass (Mr) 28,000 also stimulates colony formation by more mature erythroid precursors (CFU-E) and is therefore referred to as erythroid-potentiating activity (EPA)5. Purified EPA specifically stimulates human and murine cells of the erythroid lineage, unlike murine interleukin-3 (IL-3) which stimulates precursor cells from all haematopoietic lineages6. We report here the isolation of a complementary DNA molecular clone encoding EPA and its use in producing EPA in COS (monkey) cells and CHO (Chinese hamster ovary) cells. We also define the organization of the EPA gene in human DNA.

Purification of fetal hematopoietic progenitors and demonstration of recombinant multipotential colony-stimulating activity.

To facilitate the direct study of progenitor cell biology, we have developed a simple and efficient procedure based upon negative selection by panning to purify large numbers of committed erythroid and myeloid progenitors from human fetal liver. The nonadherent, panned cells constitute a highly enriched population of progenitor cells, containing 30.4 +/- 13.1% erythrocyte burst forming units (BFU-E), 5.5 +/- 1.9% granulocyte-macrophage colony forming units (CFU-GM), and 1.4 +/- 0.7% granulocyte-erythroid-macrophage-megakaryocyte colony forming units (CFU-GEMM) as assayed in methylcellulose cultures. These cells are morphologically immature blasts with prominent Golgi. This preparative method recovers 60-100% of the committed progenitors detectable in unfractionated fetal liver and yields 2-30 X 10(6) progenitors from each fetal liver sample, and thus provides sufficient numbers of enriched progenitors to allow direct biochemical and immunologic manipulation. Using this technique, a purified recombinant protein previously thought to have only granulocyte-macrophage colony stimulating activity (GM-CSA) is shown to have both burst promoting activity and multipotential colony stimulating activity. Progenitor purification by panning thus appears to be a simple, efficient method that should facilitate the direct study of committed hematopoietic progenitors and their differentiation.

Interleukin 4 is at 5q31 and interleukin 6 is at 7p15

SummaryDNA probes to the human interleukin 4 (IL4) and interleukin 6 (IL6) genes have been used for in situ hybridization to normal human chromosomes and Southern blot analysis of a series of mouse-human hybrid cell lines. IL4 maps to 5q31, the same location as IL5 and other haemopoietic growth factor genes. IL6 maps to 7p15. The significance of these locations is discussed.

Abstract 642: Novel EGFR inhibitory antibodies directed against EGFR mutants with potentially reduced toxicity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Non-small cell lung cancers (NSCLC) driven by ligand-independent activating mutations in epidermal growth factor receptor (EGFR) often respond to treatment with EGFR tyrosine kinase inhibitors (TKIs). About half of acquired resistance to EGFR-TKI therapy results from a secondary point mutation in the EGFR tyrosine kinase domain at amino acid position 790 (T790M). T790M mutants also display reduced sensitivity to Cetuximab treatment. We have investigated the molecular mechanism responsible for the reduced Cetuximab sensitivity and found that T790M mutant receptors primarily exist and signal as monomers. We have exploited this characteristic of the T790M mutant to isolate novel EGFR inhibitory antibodies with activities against all EGFR variants. In addition, as opposed to other EGFR inhibitory antibodies, these novel antibodies have reduced ligand binding inhibitory activity and minimal inhibitory effect on EGF induced human primary keratinocyte proliferation, suggesting a potentially reduced skin toxicity profile. The broad activity profile, combined with potentially reduced skin toxicity, suggests that these antibodies will have great potential for combinability with other therapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 642. doi:10.1158/1538-7445.AM2011-642

Chronic Myelogenous Leukemia as a Model System for Stem Cell Growth Regulation and Hematolymphoid Development

Chronic myelogenous leukemia (CML) is a disease of the pluripotent stem cell which can affect all phases of hematopoiesis. The most common clinical course shows progression from a relatively indolent chronic phase to an acute leukemic picture in the blast crisis phase. A specific chromosomal translocation called the Philadelphia chromosome or Phl has been detected in almost all patients with this clinical symptom complex at all stages of the disease. A variety of data have been accumulated which demonstrate that one molecular consequence of the Phl translocation is the production of an altered form of the abl oncogene protein by a complex splicing mechanism which joins exon sequences from chromosome 22 (called bcr) to a portion of the exons of the abl gene on chromosome 9. This altered abl protein has a high level of tyrosine kinase activity similar to the oncogenic form expressed by the Abelson murine leukemia virus. Recent cDNA cloning and sequencing results from our laboratory have demonstrated that the fused mRNA contains an extremely GC-rich segment at its 5′-end. This segment, which is referred to as gcr for guanine-cytosine rich, is probably derived from the 4.5-kb c-bcr mRNA. Understanding the regulation of this complex mRNA during the disease progression may help to decipher the pathobiology of CML and the growth regulation of the pluripotent stem cell.