Tumorigenicity associated with loss of differentiation and of response to insulin in the adipogenic cell line 1246

Summary The adipogenic cell line 1246, which grows and differentiates in defined medium, stringently requires insulin for both processes. From this cell line, insulin-independent variants were isolated and characterized. Unlike 1246 cells, the variant cell lines proliferate without insulin, have lost their differentiation ability, produce factor(s) able to replace insulin to stimulate 1246 cell growth but not differentiation and are tumorigenic. Because of these properties, this system is appropriate to examine the correlation (if any) between the loss of response to an extra-cellular factor and of ability to differentiate, and between the production of endogenous growth factor and the acquisition of tumorigenic properties. Supported by NIH grant P01 CA 37589-01 and in part by BRSG S07 RR 05863∶04, awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institute of Health. Editor's Statement This report presents evidence that the growth-promoting activity of insulin, insulin-like growth factors, or other growth factors mimicking their actions, may be separable in functional assays from differentiation-promoting activity. Data presented here also further strengthen existing hypotheses linking tumorigenicity and autocrine growth stimulation. Gordon H. Sato     

Insulin but not IGF-I is required for the maintenance of the adipose phenotype in the adipogenic cell line 1246

Summary The mouse adipogenic cell line 1246 which possesses both insulin and insulin-like growth factor I (IGF-I) receptors was used to investigate the role of IGF-I and insulin on the proliferation of adipocyte precursors and their differentiation into mature adipocytes. Results indicate that both insulin and IGF-I stimulate the proliferation of the 1246 adipocyte precursors with IGF-I being slightly more potent than insulin. Dose-response studies indicated that both polypeptides acted at physiological concentrations corresponding to binding to their own receptors. In contrast, comparison of insulin and IGF-I capacity to stimulate terminal adipose differentiation indicated that only insulin was active when added at physiological concentrations. IGF-I could not stimulate adipocyte differentiation except at supraphysiological concentrations (100 ng/ml and above) permitting its binding to the insulin receptors on 1246 cells. Time course study of expression of early and late markers of adipose differentiation indicated that the induction of markers such as adipose differentiation-related protein (ADRP), lipoprotein lipase (LPL) and fatty acid binding protein (FAB) took place even in the absence of insulin. However, the level of early and late differentiation markers decreased to a level below the one found in undifferentiated cells when cells had been maintained in the absence of insulin after differentiation had been initiated. These data indicate that although insulin is not necessary for the early onset of the adipose differentiation program, it is stringently required for the maintenance of the adipocyte phenotype and cannot be substituted by IGF-I.     

Molecular cloning of mouse PSP mRNA.

PSP is the most abundant translation product of mouse parotid glands where its production is co-ordinated with that of salivary amylase. The synthesis of these two proteins apparently is restricted to this tissue. In order to enable us to study common regulatory elements in the genes of the two proteins, double stranded cDNA, synthesized for parotid gland poly (A)+ RNA, was cloned. DNA sequencing of three clones complementary to the most abundant messenger indicated overlap and resulted in a total sequence of 867 nucleotides. Translation of this sequence revealed that at one end the amino acid sequence was the same as the N-terminal sequence of PSP. The sequence contains 60 nucleotides coding for part of or the complete signal peptide, 645 nucleotides coding for the PSP protein, and 162 nucleotides that apparently are not translated. Southern blot analysis suggests a simple structure for the PSP gene in mouse and man.     

Molecular cloning of rabbit gamma heavy chain mRNA.

A cDNA library of rabbit spleen mRNA was screened for immunoglobulin heavy chain sequences. In this paper we report the nucleotide sequence of two cDNA clones containing part of the constant region of the rabbit gamma heavy chain mRNA. The sequence encodes part of the CH2 domain (amino acids 268 to 340), the entire CH3 domain (amino acids 341 to 447) and the 3' untranslated region. This nucleotide sequence has been compared to the corresponding sequences of mouse gamma 1, gamma 2a and gamma 2b genes. The homologies between rabbit gamma chain gene sequence and each of the mouse gamma chain gene sequences are of the same magnitude order. This comparison shows that the CH2 domains are more homologous to each other than CH3 domains or 3' untranslated sequences. The presence of species specific nucleotide positions suggests that mouse gamma chain genes could have evolved from a common ancestor shortly after the mouse-rabbit species separation. Genomic blot analysis of rabbit liver DNA with the rabbit C gamma probes shows a limited number of related sequences, with little restriction site polymorphism between individual rabbits.     

Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line.

Macrophages activated by exposure to cytokines and/or to endotoxin produce nitric oxide (NO.), a free radical that is a mediator of the host response to infection. Activation induces the expression of nitric oxide synthase, the enzyme that catalyzes formation of NO. from L-arginine and molecular oxygen. We report the cloning of a cDNA encoding the inducible nitric oxide synthase from a murine macrophage cell line, RAW264.7, exposed to interferon-gamma and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO.. Nitric production is blocked by the enzyme inhibitor, NG-monomethylarginine, and is independent of calcium/calmodulin. RAW264.7 cells demonstrate rapid accumulation of the nitric oxide synthase-encoding mRNAs upon activation. Comparison of the deduced amino acid sequence to the calcium/calmodulin-dependent nitric oxide synthase previously purified (Bredt, D. S., and Synder, S.H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685) and cloned (Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Synder, S. H. (1991) nature 351, 714-718) from rat brain identifies shared binding sites for the cofactors NADPH and flavins in the C-terminal half of both proteins and an additional conserved region near the N terminus that may recognize L-arginine and/or contribute to the active site.     

Molecular cloning and permanent expression in a neuroblastoma cell line of a fast inactivating potassium channel from bovine adrenal medulla.

Using a cDNA library from bovine adrenal medulla, we have isolated cDNAs coding for a potassium channel. These cDNAs encode a 660-amino acid protein that has a molecular weight of 73,288 kDa and no amino-terminal signal peptide. We have called it BAK4. Analysis of its sequence reveals close similarity (94% homology) with a recently described potassium channel from rat brain (RCK4) and heart (RHK1). Neuroblastoma cells (Neuro-2a cell line) were stably transfected with BAK4 DNA. Expression of the DNA was under the control of a heat-shock promoter. Several clones, that were isolated by neomycin resistance selection, had integrated the plasmid DNA in a stable form. Upon heat induction, these cells produced BAK4 RNA and a potassium outward current, not present in control non-transfected cells. The current, which was transient and decayed markedly during the duration of 200 ms-pulses, can be described as a Ik(A) potassium current. The expression of these types of channels in brain (RCK4,RHK1), heart (RHK1) and adrenal medulla (BAK4) suggest their possible implication in important functions for the cell.     

Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line.

Molecular analysis of Saccharomyces cerevisiae secretion mutants has led to the identification of two Ras-like GTP-binding proteins, Ypt1p and Sec4p, which are essential for transport along the exocytic route. To study the regulation of membrane traffic in epithelial cells, a set of 11 clones encoding proteins similar to the YPT1/SEC4 products were isolated from an MDCK (Madin-Darby canine kidney) cell cDNA library. Four of these proteins, Rab8, -9, -10, and -11, are novel members of this subfamily of Ras-like proteins, and two of them are closely related to Ypt1p and Sec4p. The ratio of the number of clones isolated over the total number screened reveals a high level of complexity for this subfamily of GTP-binding proteins. This diversity supports their proposed function in controlling different steps in membrane traffic.     

Identification of insulin receptors on the insulin-independent variant 1246-3A cell line

1246-3A cell line is an insulin-independent variant isolated from the adipogenic cell line 1246 which can proliferate in the absence of insulin, has lost the ability to differentiate, and secretes an insulin-related factor called IRF similar to pancreatic insulin and different from IGFs. In contrast, the parent adipogenic cell line 1246 is dependent on the presence of insulin to proliferate and differentiate in defined medium. In the present paper, we examined if the loss of response to insulin observed for 1246-3A cells was accompanied by alterations in the insulin receptor properties. Insulin binding and tyrosine kinase activity of insulin receptors isolated from 1246-3A cells and from the parent cell line 1246 were measured; 125I-insulin binding to intact cells was 75% lower for the 1246-3A cells than for the 1246 cells. This was due to a decrease in receptor number without major change in receptor affinity. However, when the cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed for both cell lines. Down regulation of insulin receptors by insulin occurred in a dose-dependent fashion, which was similar for both cell lines. Phosphorylation experiments were performed by incubation of the partially purified insulin receptor with insulin and [gamma-32P]ATP. They indicated that insulin stimulated phosphorylation of the 95-kDa molecular weight beta subunit of the receptor, in a similar fashion for both cell types. These data suggest that the insulin-independent cell line 1246-3A does not possess a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties and that the loss of response to insulin can be attributed to the fact the 1246-3A cells secrete IRF which bind to cell surface receptors and stimulate their proliferation.     

Molecular cloning and functional expression of a Talaromyces emersonii derived alpha-amylase encoding genetic determinant in a human cell line

Summary A cDNA fragment encoding a Talaromyces emersonii acid stable alpha-amylase has been cloned into a mammalian cell expression vector system. When human HeLa cells were transformed with this DNA, functional enzyme could be detected in extracellular media and cell lysates derived from stable transformants. Expression of the T. emersonii derived cDNA was verified by northern dot blotting hybridisation analysis of mRNA extracted from transformants, using the labelled amylase encoding cDNA determinant as a probe. Results obtained suggest that expression of the fungus derived alpha-amylase in the transformed HeLa cells is glucocorticoid dependent.     

Molecular cloning and expression of two distinct cDNA-encoding heparan sulfate proteoglycan core proteins from a rat endothelial cell line.

The cloned rat fat pad endothelial cell (RFP-EC) line synthesizes anticoagulantly active heparan sulfate proteoglycans (HSPGact) and anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact), both of which exhibit 25-, 30-, and 50-kDa core proteins of extremely similar structure. The primary sequences of internal peptides obtained from HSPGinact core proteins and the NH2-terminal sequence analyses of the 25-kDa component from the HSPGinact core proteins demonstrate that the 30-kDa component is a previously unidentified species, designated as ryudocan, with the 25-kDa component representing a proteolytic degradation product, while the 50-kDa component is the rat homolog of syndecan (Saunders, S. Jalkanen, M., O'Farrell, S., and Bernfield, M. (1989) J. Cell Biol. 108, 1547-1556). Specific oligonucleotide probes were obtained for ryudocan and syndecan by polymerase chain reaction, and the corresponding cDNAs were isolated from a RFP-EC library. The cDNAs encode type I integral membrane proteins of 202 and 313 amino acids, respectively, which have homologous transmembrane and intracellular domains but very distinct extracellular regions. In particular, ryudocan exhibits only three potential glycosaminoglycan attachment sites within the extracellular region while syndecan has five glycosaminoglycan attachment sites within the same domain. Both species are expressed in RFP-EC lines, primary rat aortic smooth muscle cells and primary rat skin fibroblast cells. The levels of ryudocan and syndecan mRNA were measured by quantitative polymerase chain reaction in primary microvascular endothelial cells and closely associated non-endothelial cells isolated by cell sorting. Ryudocan and syndecan mRNAs were abundantly expressed in both populations representing about 0.1-0.5% of mRNA.     

Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line

Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the endoplasmic reticulum and Golgi lumen. In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis. The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterized. In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning using a Chinese hamster ovary cell line (CHO-Lec8) deficient in UDP-galactose transport. The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs. Despite this low overall identity, the two proteins clearly belong to the same gene family. Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in vitro NST assay. The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate, and xylulose 5-phosphate. Also, the latter proteins are members of the same family, which therefore has been named the NST/triose-phosphate transporter family.     

Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors.

We wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first we located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10(5) thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.     

Molecular cloning and characterization of the mRNA for cyclin from sea urchin eggs.

We have isolated a cDNA clone encoding sea urchin cyclin and determined its sequence. It contains a single open reading frame of 409 amino acids which shows homology with clam cyclins. RNA transcribed in vitro from this sequence was efficiently translated in reticulocyte lysates, yielding full-length cyclin. Injection of nanogram amounts of this synthetic mRNA into Xenopus oocytes caused them to mature more rapidly than with progesterone treatment. The sea urchin cyclin underwent two posttranslational modifications in the Xenopus oocytes during maturation. The first occurred at about the time that maturation became cycloheximide-resistant, when a small apparent increase in the molecular weight of cyclin was observed. The second modification involved destruction of the cyclin at about the time of white spot appearance, just as would have occurred at the metaphase/anaphase transition in the natural environment of a cleaving sea urchin embryo.