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The mammalian shc gene encodes two overlapping proteins of 46 and 52 kDa, each with a C-terminal Src homology 2 (SH2) domain and an N-terminal glycine/proline-rich sequence, that induce malignant transformation when overexpressed in mouse fibroblasts. p46shc, p52shc, and an additional 66-kDa shc gene product become highly tyrosine phosphorylated in Rat-2 cells transformed by the v-src or v-fps oncogene. Experiments using temperature-sensitive v-src and v-fps mutants indicate that Shc tyrosine phosphorylation is rapidly induced upon activation of the v-Src or v-Fps tyrosine kinases. These results suggest that Shc proteins may be directly phosphorylated by the v-Src and v-Fps oncoproteins in vivo. In cells transformed by v-src or v-fps, or in normal cells stimulated with epidermal growth factor, Shc proteins complex with a poorly phosphorylated 23-kDa polypeptide (p23). Activated tyrosine kinases therefore regulate the association of Shc proteins with p23 and may thereby control the stimulation of an Shc-mediated signal transduction pathway. The efficient phosphorylation of Shc proteins and the apparent induction of their p23-binding activity in v-src- and v-fps-transformed cells are consistent with the proposition that the SH2-containing Shc polypeptides are biologically relevant substrates of the oncogenic v-Src and v-Fps tyrosine kinases.

The lineage and clonality of Hodgkin's disease (HD) were investigated by analyzing the organization of the immunoglobulin and T-cell receptor beta-chain (T beta) gene loci in 18 cases of HD, and for comparison, in a panel of 103 cases of B- and T-cell non-Hodgkin's lymphomas (NHLs) and lymphoid leukemias (LLs). Sizable clonal B- or T-cell populations, representing greater than or equal to 10% of the pathologic sample, were readily detectable by immunogenotypic analysis in all 103 NHLs and LLs but not in any of the 18 cases of HD. However, extremely minor clonal populations (less than or equal to 1%) were detectable in 3 of 18 cases of HD. We demonstrated that these minor clonal populations do not correspond to Reed-Sternberg (RS) cells since clonal immunoglobulin or T beta gene rearrangements are not detectable in cases of HD containing greater than 25% RS cells. The number of RS cells present in these samples appeared to correlate directly with the pattern of gene rearrangements characteristic of polyclonal T cells. These studies demonstrate that Southern blot hybridization analysis for clonal immunoglobulin and T beta gene rearrangements represents an accurate, objective tool in the differential diagnosis between HD and NHL; that HD is predominantly composed of polyclonal B and T cells; that minor clonal B- or T-cell populations unrelated to RS cells occasionally can be found in HD; and that RS cells do not represent clonal B- or T-cell expansions. Finally, our data preliminarily suggest that RS cells may represent polyclonal T-cell populations.

In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic origin. These observations prompted our search for additional EH-containing proteins in mammalian cells. Using an EH domain-specific probe derived from the eps15 cDNA, we cloned and characterized a cDNA encoding an EH-containing protein with overall similarity to Eps15; we designated this protein Eps15r (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins.

We have examined the position of the chromosomal breakpoint relative to the human c-myc gene (MYC) and the presence of other structural alterations of the same locus in 19 fresh samples of Burkitt lymphoma (BL) and 13 BL-derived cell lines. This panel includes the two pathogenetic forms of BL: the endemic (African-type) BL (eBL) and sporadic (American-type) BL (sBL). In all cases tested, including fresh samples and cell lines, structural alterations of the 5' portion of the gene were detected, suggesting that they may be necessary for c-myc activation. However, the site of chromosomal breakpoint and the type of structural alterations differ in eBL and sBL. In 16 of 18 sBL cases, chromosomal translocation truncates the gene within a 5' region that includes the first intron, the first exon, and 5' flanking sequences. Conversely, in 14 of 14 eBL samples, the chromosomal breakpoint is located outside the c-myc locus, yet the same 5' sequences are affected by several mutations identifiable by restriction enzyme polymorphisms. Different genetic mechanisms may therefore be involved in chromosomal translocation/c-myc activation, and these differences may be a function of differences in the stage of differentiation of eBL versus sBL.

An increasingly large number of proteins involved in signal transduction have been identified in recent years and shown to control different steps of cell survival, proliferation, and differentiation. Among the genes recently identified at the tip of the long arm of the human X chromosome, a novel gene, C1, encodes a protein that appears to represent a newly discovered member of the group of signaling proteins involved in regulation of the small GTP binding proteins of the ras superfamily. The protein encoded by C1, p115, is synthesized predominantly in cells of hematopoietic origin. It is characterized by two regions of similarity to motifs present in known proteins: GAP and SH3 homologous regions. Its localization in a narrow cytoplasmic region just below the plasma membrane and its inhibitory effect on stress fiber organization indicate that p115 may down regulate rho-like GTPases in hematopoietic cells.

Ig gene rearrangements represent markers of lineage, clonality, and differentiation of B cells, allowing a molecular diagnosis and immunogenotypic classification of B-cell neoplasms. We sought to apply a similar approach to the study of T-cell populations by analyzing rearrangements of the T-cell receptor beta-chain (T beta) gene. Our analysis, by Southern blotting hybridization using T beta-specific probes of DNAs from polyclonal T cells and from 12 T-cell tumors, indicates that T beta gene rearrangement patterns can be used as markers of (i) lineage, allowing the identification of polyclonal T-cell populations, and (ii) clonality, allowing the detection of monoclonal T-cell tumors. In addition, our data indicate that T beta gene rearrangements represent early and general markers of T-cell differentiation since they are detectable in histologically different tumors at all stages of T-cell development. The ability to determine lineage, clonality, and stage of differentiation has significant implications for future experimental and clinical studies on normal and neoplastic T cells.

Human leukemia cells in culture (HL-60) synthesize and secrete proteins that are recognized by antiserum to human platelet-derived growth factor (PDGF). The molecular mass of the intracellular proteins immunoprecipitated by PDGF antiserum ranged from 34 kDa to 240 kDa. PDGF-related proteins were also identified in the conditioned medium of the cells. Several of these immunoprecipitated proteins were glycosylated. A single protein of 46 kDa was immunoprecipitated from the cell-free translation products of mRNA obtained from the leukemia cells. Antiserum to the C but not to the N terminus of the predicted amino acid sequence of the transforming protein p28sis/PDGF-2 also immunoprecipitated proteins secreted by the HL-60 cells. These findings provide a direct demonstration for the synthesis and secretion of PDGF-like proteins by leukemia cells in culture. These proteins do not appear to be coded by the known c-sis/PDGF-2 locus since no sis mRNA was detectable in the HL-60 cells.

Two chimeric genes, PML-RAR alpha and RAR alpha-PML, are formed as a consequence of the acute promyelocytic leukemia (APL)-specific reciprocal translocation of chromosomes 15 and 17 [t(15;17)]. PML-RAR alpha is expressed as a fusion protein. We investigated the organization and expression pattern of the RAR alpha-PML gene in a series of APL patients representative of the molecular heterogeneity of the t(15;17) and found (i) two types of RAR alpha-PML mRNA junctions (RAR alpha exon 2/PML exon 4 or RAR alpha exon 2/PML exon 7) that maintain the RAR alpha and PML longest open reading frames aligned and are the result of chromosome 15 breaking at two different sites; and (ii) 10 different RAR alpha-PML fusion transcripts that differ for the assembly of their PML coding exons. A RAR alpha-PML transcript was present in most, but not all, APL patients.

Immunoprecipitation of proteins extracted from metabolically labeled human glioblastoma and fibrosarcoma cells with antiserum to platelet-derived growth factor (PDGF) showed that these cells express and secrete proteins that are recognized specifically by the antiserum. The molecular masses of immunoprecipitated proteins in the lysates of the malignant cells ranged from 16 kDa to 140 kDa. Both cell lines secreted a 31-kDa polypeptide with structural, immunological, and biological properties similar to those of human PDGF. These cell lines were shown to synthesize a 4.4-kb mRNA that contained sequences from all the six currently identified exons of the human c-sis gene. These data suggest that the PDGF-like proteins in the two mesenchyme-derived transformed cells are encoded at least in part by the c-sis locus.