We have used polyclonal anti-synthetic peptide serum to study the role of retinoblastoma gene (RB) inactivation in a variety of human tumor cell lines. Our analysis indicates that inactivation of the RB protein, p105-Rb, is universal in retinoblastoma cells, vindicating the predictions of the Knudson "two-hit" hypothesis. In addition, our analysis has shown that inactivations of the RB gene are nearly as frequent in a more common human tumor, small cell lung carcinoma. One-third of bladder carcinomas surveyed also carry altered or absent p105-Rb. Other human tumors by contrast demonstrate only infrequent inactivation of the RB gene. These results suggest that inactivation of the RB gene is a critical step in the pathogenesis of a subset of human tumors.The recent isolation of molecular clones of the retinoblastoma gene (RB) has made it possible to evaluate the role of RB gene inactivation in the genesis of a variety of human tumors (1-3). These molecular analyses have confirmed and extended earlier karyotypic and genetic studies that had implied that both copies ofthis gene suffer inactivation during the formation of retinoblastomas and several types of sarcoma (4-10). Since the inactivation ofRB function appears to trigger tumorigenesis, the RB gene has been termed a "tumor suppressor" gene, whose expression is required to constrain normal cellular proliferation.Analysis of the protein product encoded by the RB gene, p105-Rb, has shown it to be a nuclear phosphoprotein having an affinity for DNA (11,12 . This complex formation is apparently central to the ability of these oncoproteins to transform primary cells (14,17).In the initial studies designed to detect inactivation of chromosomal copies of the RB gene, cloned segments of the RB gene were used as probes in Southern blot analysis of tumor DNAs (1)(2)(3)(18)(19)(20)(21) MATERIALS AND METHODSPreparation of Cell Lysates and Immunoprecipitation of the RB Protein from Human Tumor Cells. Tumor cell cultures were incubated for 3-5 hr with [35S~methionine, and cell lysates were prepared as described (13). Immunoprecipitations were performed with rabbit serum no. 147, 144, or 140 (13), and precipitates were resolved by electrophoresis for 15 hr on SDS/polyacrylamide gels, processed for fluorography, and exposed for 1-3 days at -70'C. Bladder, colon, and breast carcinoma cultures were acquired from the American Type Culture Collection as were two retinoblastoma cultures (Y79 and WERI-1). The derivation ofall other retinoblastoma cultures and small cell lung carcinoma (SCLC) cultures has been described (1,23,24). Melanoma tumor cells were a kind gift of Nick Dracopoli of the Massachusetts Institute of Technology.Amplification ofTumor Cell mRNA by Using the Polymerase Chain Reaction (PCR) Technique and DNA Sequence Analysis. RB mRNA was specifically amplified by using oligonucleotide primers from the RB cDNA sequence and the PCR as described (12). Amplified fragments were cloned into M13 and sequenced on both strands by the dideoxy chain termination techniq...

Retinoblastoma is a childhood tumor that can arise because of mutant alleles acquired as somatic or germinal mutations. The mutant allele can be carried in the germ line. The mutations creating these alleles act by inactivating copies of a recessive oncogene located within band q14 of chromosome 13 and termed the RB1 locus. We have reported isolation of a cDNA fragment that recognizes chromosomal sequences possessing many of the attributes of the retinoblastoma gene associated with the RB1 locus. We now report that this segment is additionally the target of somatic mutations in mesenchymal tumors among patients having no apparent predisposition to retinoblastoma and no previous evidence of retinoblastoma. These tumors provide additional evidence that the cloned sequences are representative of a gene that is a frequent target of inactivation during tumorigenesis. Sequence analysis of this cDNA provides little insight into its normal functional role.

The expression of gp140TRK-A mRNA correlates with distinct biologic and clinical subsets of neuroblastoma, which suggests a role for the high-affinity nerve growth factor receptors in determining the phenotype of neuroblastoma. The absence of gp140TRK-A mRNA expression, whether or not the N-myc proto-oncogene is amplified, is associated with tumor progression.

The human trkA cDNA was transfected into a malignant human neuroblastoma (NB) cell line (HTLA230) to investigate its role in NB growth and differentiation. This cell line lacks expression of both endogenous trkA and gp75NGFR genes. Transfectants expressing the trkA mRNA and surface-bound receptors transcriptionally activate immediate-early genes (c-fos, c-jun, and jun-B) following nerve growth factor (NGF) stimulation. NGF treatment induces growth arrest as well as down-regulation of the amplified N-myc oncogene. Genes selectively expressed in mature neurons (SCG-10, ret proto-oncogene, GAP-43, etc.) are transcriptionally activated, and neurite outgrowth further demonstrates differentiation of transfectants following NGF stimulation. trkA-expressing NB cells remain tumorigenic in nude mice; however, subcutaneous treatment of tumor-bearing mice with NGF induces Schwannian and neuronal cell differentiation similar to the induction seen in human ganglioneuroblastomas. Thus, trkA expression in HTLA230 cells is sufficient to generate a functional NGF receptor complex that leads to growth-arrested and differentiated NB cells in vitro and in vivo in the presence of NGF. Hence, NGF may play a crucial role in NB cell differentiation and regression in vivo.

We have isolated a cDNA clone of the murine homologue of the human retinoblastoma (Rb) susceptibility gene. DNA sequence analysis reveals a high degree of conservation with the human Rb sequence, both in the coding and in the noncoding regions. The predicted amino acid sequence of the mouse Rb protein shows 91% identity to that of the human protein. Both proteins were found to contain a peptide sequence reminiscent of a leucine-repeat motif ("leucine-zipper") that is also found in the myc, fos, and jun oncogenes. Synthetic peptide antiserum directed against a portion of the mouse Rb protein detects three proteins of 104-110 kDa in cells that were transiently transfected with a mouse Rb gene expression construct. In the mouse embryo the expression of Rb mRNA was ubiquitous, with maximal expression being observed around 13 days of gestation. In the embryo, the highest level of expression was observed in liver and brain. In contrast, the Rb gene was found to be expressed at a very low level in adult mouse liver with high levels being found in lung, thymus, and spleen. A shorter Rb transcript was detected in mouse testes.

The p75NTR neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75NTR, many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75NTR (p75NTR-FX) allele made by flanking exons 4–6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75NTR/Wnt1-Cre mutants and conventional p75NTR null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75NTR in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75NTR mutants. This novel conditional p75NTR allele will offer new opportunities to investigate the role of p75NTR in specific tissues and cells.

We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels. The cells were cultured for 1 d in serum-containing medium and then for up to 25 d in chemically defined medium. In some experiments, epithelial monolayers on gels were mechanically freed from the dishes on day 2 or 5. These gels retracted and formed floating collagen gels. On attached collagen gels, flat monolayers of a single cell type developed within a few days. The cells synthesized DNA until the achievement of confluence but did not accumulate milk proteins. No morphological changes were induced by prolactin (PRL). On floating gels, two cell types appeared in the absence of cell proliferation. The cells in direct contact with the medium became cuboidal and developed intracellular organelles typical of secretory cells. PRL-induced lipogenesis, resulting in large fat droplets filling the apical cytoplasm and accumulation of casein and cx-lactalbumin in vesicles surrounding the fat droplets. We detected transferrin in the presence or absence of PRL intracellularly in small vesicles but also in the collagen matrix in contact with the cell layer. The second cell type, rich in microfilaments and reminiscent of the myoepithelial cells, was situated between the secretory cell layer and the collagen matrix. In embedding gels, the cells formed hollow ductlike structures, which grew continuously in size. Secretory cells formed typical lumina distended by secretory products. We found few microfilament-rich cells in contact with the collagen gels. Storage and secretion of fat, caseins and ~x-lactalbumin required the presence of PRL, whereas the accumulation and vectorial discharge of transferrin was prolactin independent. There was no differentiation gradient between the tip and the center of the outgrowth, since DNA synthesis and milk protein storage were random along the tubular structures.These results indicate that establishment of functional polarity and induction of cytodifferentiation are influenced by the nature of the interaction of the cells with the collagen structure. The morphological differentiation in turn plays an important role in the synthesis, storage, and secretion of fat and milk proteins.Onset of gestation is reflected in the mammary gland by the resumption of epithelial cell proliferation with a characteristic lobulo-alveolar outgrowth into fat tissue previously free of ductal penetration. The cell growth of the mammary gland is

Retinoblastoma, an intraocular tumor that occurs in children, has long been regarded, on the basis of morphological criteria, as a malignancy of the photoreceptor cell lineage. Here it is shown that when this tumor is grown in vitro, the cells express highly specialized photoreceptor cell genes. Transcripts for the transducin alpha subunit, TC alpha, which is specific to the cone cell, as well as transcripts for the red or green cone cell photopigment, were found in seven out of seven low-passage retinoblastoma cell lines. No marker genes specific to rod cell were expressed, suggesting that retinoblastoma has a cone cell lineage.