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The development and maintenance of the nervous system depends on proteins known as neurotrophic factors. Although the prototypical neurotrophic factor, nerve growth factor (NGF), has been intensively studied for decades, the discovery and characterization of additional such factors has been impeded by their low abundance. Sequence homologies between NGF and the recently cloned brain-derived neurotrophic factor (BDNF) were used to design a strategy that has now resulted in the cloning of a gene encoding a novel neurotrophic factor, termed neurotrophin-3 (NT-3). The distribution of NT-3 messenger RNA and its biological activity on a variety of neuronal populations clearly distinguish NT-3 from NGF and BDNF, and provide compelling evidence that NT-3 is an authentic neurotrophic factor that has its own characteristic role in vivo.

The availability of relatively large amounts of nerve growth factor (NGF) has allowed extensive in vitro and in vivo characterization of the neuronal specificity of this neurotrophic factor. The restricted neuronal specificity of NGF (sympathetic neurons, neural crest-derived sensory neurons, basal forebrain cholinergic neurons) has long predicted the existence of other neurotrophic factors possessing different neuronal specificities. Whereas there have been many reports of "activities" distinct from NGF, full characterization of such molecules has been hampered by their extremely low abundance. The recent molecular cloning of brain-derived neurotrophic factor (BDNF) revealed that this protein is closely related to NGF and suggested that these two factors might be members of an even larger gene family. A PCR cloning strategy based on homologies between NGF and BDNF has allowed us to identify and clone a third member of the NGF family which we have termed neurotrophin-3 (NT-3). The establishment of suitable expression systems has now made available sufficient quantities of these proteins to allow us to begin to establish the neuronal specificity of each member of the neurotrophin family, and the role of each in development, maintenance and repair of the PNS and CNS. Using primary cultures of various PNS and CNS regions of the developing chick and rat, and Northern blot analysis, we describe novel neuronal specificities of BDNF, NT-3 and an unrelated neurotrophic factor-ciliary neurotrophic factor (CNTF).

The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.

Variability in the phenotype of cells comprising individual tumours is a striking feature of animal and human cancer and is generally referred to as tumour heterogeneity. Studies of clonally derived cell populations from tumours that originated presumably from a single transformed cell have shown that tumours are made up of cells that differ in a variety of traits, including drug resistance, antigen expression and metastatic potential. The origin and maintenance of tumour heterogeneity are unclear, but mutational and epigenetic mechanisms are thought to be involved. Here we report the results of a search for transforming genes in human melanoma which have raised the possibility that ras gene activation follows the same variable pattern as other traits involved in tumour heterogeneity. DNA from 4 of 30 melanoma cell lines yielded transforming ras genes in the NIH/3T3 assay. Of five cell lines originating from separate metastatic deposits of a single patient, only one contained activated ras, indicating heterogeneity in ras activation in this case and suggesting that ras activation was not involved in tumour initiation or maintenance in this patient.

The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.

Antisera were prepared in mice, rats and rabbits by immunization with peptides corresponding to regions of highest variability, located near the C-termini of four ras proteins. Two of these, H-ras (171-189) and K-rasB (171-186), react uniquely with H-ras and K-rasB gene products in immunoblots and immunoprecipitation reactions. Affinity-purified rabbit H-ras (171-189) antibody detects H-ras p21 in tissue culture cells and in tissue sections. Epithelial cells in normal mouse skin and cells in papillomas and carcinomas, in a mouse model system of chemical carcinogenesis in which mutational activation of H-ras occurs with high frequency, express high levels of H-ras p21 protein. These results suggest an hypothesis to explain the mechanism and preferential activation of particular ras loci in certain neoplasia.

The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.

The expression of normal and mutant ras genes in human acute leukemias was assessed by the direct analysis of p21ras polypeptides, using immunoprecipitation with monoclonal antibodies. High-resolution two-dimensional gel electrophoresis permits the identification of a wide array of activated ras alleles encoding proteins with single amino acid substitutions at any of several positions. The products of three ras genes, H-ras, N-ras, and K-ras, were detected in each of 33 specimens of fresh leukemic cells. The normal K-ras and N-ras polypeptides were substantially more abundant than H-ras p21 in all samples. In over three-fourths of the cases the total amount of p21ras exceeded that seen in control hematopoietic cell lines. The level of ras expression did not correlate simply with clinical parameters, although the two samples with the most abundant p21ras were obtained from patients with relapsed T-cell acute lymphocytic leukemia (ALL). Abnormal p21ras, consistent with oncogenic activation, was found in eight patients. Six of 11 samples from acute myelocytic leukemia (AML) patients displayed a mutant N-ras p21, while only one of 20 ALL specimens had abnormal N-ras, and one had a mutant H-ras. In every case the mutant protein comprised a minority of total p21ras. In two T-cell ALL cell lines both normal and activated N-ras gene products were expressed at equal levels. By contrast, in five fresh AML samples the abnormal N-ras protein was several-fold less abundant than the normal N-ras p21. This finding implies that only a proportion of leukemic cells in an individual patient may carry the mutant ras oncogene.

We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides.

Human hematopoietic malignancies provide an excellent model for the study of the activity of cellular oncogenes in a context of known defects in cell proliferation and differentiation. A flow cytometric immunofluorescence assay was developed to quantitate the expression of the cellular ras oncogenes in relation to the cell cycle in individual leukemic cells. Specific binding of a monoclonal antibody to the 21-kd protein (p21ras) encoded by the Ha-ras, Ki-ras, and N-ras genes was measured by flow cytometry and confirmed by fluorescence microscopy. P21ras was detected in 416B, a murine hematopoietic precursor cell characterized by a high level of Ki-ras expression, and in the human leukemic cell lines P-12 and KG-1. The presence of p21ras in the cell lines was also shown by immunoprecipitation. Cellular DNA content was determined simultaneously to define cell cycle phases. There was an equal distribution of p21ras in G1, S, and G2M, with considerable heterogeneity of ras gene expression in the G1 compartment. The assay allows oncogene expression to be studied in populations of intact single cells in which cell heterogeneity is maintained, requires very few cells per sample, and directly correlates oncogene expression to cell kinetic data.

We report the clinical evaluation of an improved DNA probe assay for the characteristic genetic marker of human CML, observed by cytogenetics and designated the Philadelphia chromosome (Ph1). The Ph1 chromosome results from the fusion of c-abl proto-oncogene sequences from chromosome 9 to phl gene sequence on chromosome 22. (The phl gene is often referred to as bcr. However, for clarity we prefer to reserve the designation "bcr" for the region within the phl gene in which translocation breakpoints have been found to occur. We also find it useful to distinguish between two such regions in phl, bcr-210 and bcr-190, named after the 210- and 190-kDa phl/abl fusion proteins resulting from translocations with breakpoints in the respective regions. We refer to the corresponding chromosomal translocations as Ph1(bcr-210) and Ph1(bcr-190).) DNA, extracted from peripheral blood (PB) or bone marrow (BM) and digested with restriction endonuclease BglII, is hybridized with a probe (phl/bcr-3) spanning a breakpoint cluster region within phl. Rearrangements are revealed by the presence of one or two novel junction fragments. Clinical specimens from leukemic patients with active disease were compared by cytogenetic and DNA probe analysis at seven centers in the United States and Europe. The probe assay identified the phl rearrangement in 190 of 191 cases of Ph1-positive CML, as well as in 12 of 27 clinically diagnosed CML specimens lacking a typical Ph1 chromosome. DNA rearrangements also were seen in two of six cases of Ph1-positive ALL. No false positive results were obtained among 93 non-leukemic controls. Mixing experiments showed that the DNA probe assay can detect as few as 1% leukemic cells in a specimen. A preliminary study of CML patients in remission after allogeneic BM transplantation revealed a small fraction of residual Ph1-positive leukemic cells in a significant number of such patients.