Simian Virus 40-transformed Human Fibroblasts Research Articles

Bioactive Dibenzylbutyrolactone and Dibenzylbutanediol Lignans from Peperomia duclouxii

Six new dibenzylbutyrolactone (6-11) and two new dibenzylbutanediol lignans (12, 13) were obtained from Peperomia duclouxii. The structures were elucidated mainly by the analysis of NMR and MS data. The anticancer activity against a normal (WI-38) and a simian virus 40-transformed human lung fibroblast cell (VA-13) and a hepatoma G2 cell (HepG2) and the MDR reversal activity of the isolated compounds were examined. Compound 7 showed moderate inhibitory activity against VA-13 and HepG2 with IC(50) values of 23.2 and 26.4 microM, respectively. Compound 2 inhibited the growth of HepG2 cells with an IC(50) of 42.8 microM. Compounds 2 and 13 exhibited stronger MDR reversal activity than verapamil, at 25 and 2.5 microg/mL, respectively, and 4, 5, and 7 showed comparable activity with verapamil, at 25, 25, and 2.5 microg/mL, respectively.

Overexpression of Diacylglycerol Acyltransferase-1 Reduces Phospholipid Synthesis, Proliferation, and Invasiveness in Simian Virus 40-transformed Human Lung Fibroblasts

Diacylglycerol (DAG) is a versatile molecule that participates as substrate in the synthesis of structural and energetic lipids, and acts as the physiological signal that activates protein kinase C. Diacylglycerol acyltransferase (DGAT), the last committed enzyme in triacylglycerol synthesis, could potentially regulate the content and use of both signaling and glycerolipid substrate DAG by converting it into triacylglycerol. To test this hypothesis, we stably overexpressed the DGAT1 mouse gene in human lung SV40-transformed fibroblasts (DGAT cells), which contains high levels of DAG. DGAT cells exhibited a 3.9-fold higher DGAT activity and a 3.2-fold increase in triacylglycerol content, whereas DAG and phosphatidylcholine decreased by 70 and 20%, respectively, compared with empty vector-transfected SV40 cells (Control cells). Both acylation and de novo synthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were reduced by 30-40% in DGAT cells compared with controls, suggesting that DGAT used substrates for triacylglycerol synthesis that had originally been destined to produce phospholipids. The incorporation of [14C]DAG and [14C]fatty acids released from plasma membrane by additions of either phospholipase C or phospholipase A2 into triacylglycerol was increased by 6.2- and 2.8-fold, respectively, in DGAT cells compared with control cells, indicating that DGAT can attenuate signaling lipids. Finally, DGAT overexpression reversed the neoplastic phenotype because it dramatically reduced the cell growth rate and suppressed the anchorage-independent growth of the SV40 cells. These results strongly support the view that DGAT participates in the regulation of membrane lipid synthesis and lipid signaling, thereby playing an important role in modulating cell growth properties.

Inhibition of the proteasome activity, a novel mechanism associated with the tumor cell apoptosis-inducing ability of genistein

Epidemiological studies have suggested that increased soy consumption is associated with reduced cancer occurrence. Genistein, a soy isoflavone, has been reported to inhibit the growth of human tumor cells although the involved molecular mechanisms are not clearly defined. Here we report that genistein inhibits the proteasomal chymotrypsin-like activity in vitro and in vivo. Computational docking studies suggest that the interaction of genistein with the proteasomal β5 subunit is responsible for inhibition of the chymotrypsin-like activity. Inhibition of the proteasome by genistein in prostate cancer LNCaP and breast cancer MCF-7 cells is associated with accumulation of ubiquitinated proteins and three known proteasome target proteins, the cyclin-dependent kinase inhibitor p27 Kip1, inhibitor of nuclear factor-κB (IκB-α), and the pro-apoptotic protein Bax. Genistein-mediated proteasome inhibition was accompanied by induction of apoptosis in these solid tumor cells. Finally, genistein induced proteasome inhibition and apoptosis selectively in simian virus 40-transformed human fibroblasts, but not in their parental normal counterpart. Our results suggest that the proteasome is a potential target of genistein in human tumor cells and that inhibition of the proteasome activity by genistein might contribute to its cancer-preventive properties.

Reverse genetic system for the analysis of parvovirus telomeres reveals interactions between transcription factor binding sites in the hairpin stem.

The left-hand or 3'-terminal hairpin of minute virus of mice (MVM) contains sequence elements essential for both viral DNA replication at the left-hand origin (oriL) and for the modulation of the P4 promoter, from which the viral nonstructural gene cassette is transcribed. This hairpin sequence has proven difficult to manipulate in the context of the viral genome. Here we describe a system for generating mutant viruses using synthetic hairpin oligonucleotides and a truncated form of the infectious clone. This allows manipulation of the sequence of the left-hand hairpin and examination of the effects in the context of the viral life cycle. We have confirmed the requirement for a functional parvovirus initiation factor (PIF) binding site and determined that an optimized PIF binding site, with 6 bases between the half-sites, was actually detrimental to viral growth. The distal PIF half-site overlaps a cyclic AMP-responsive element (CRE), which was shown to play an important role in initiating infection, particularly in 324K simian virus 40-transformed human fibroblasts. Interestingly, reducing the spacing of the PIF half-sites, and thus the affinity of the binding site for PIF, increased viral fitness relative to wild type in 324K cells, but not in murine A9 cells. These results indicate that the relative importance of factor binding to the CRE and PIF sites during the establishment of an infection differs markedly between these two host cells and suggest that the suboptimal spacing of PIF half-sites found in wild-type virus represents a necessary reduction in the affinity of the PIF interaction in favor of CRE function.

DNA Repair Patch-mediated Double Strand DNA Break Formation in Human Cells

DNA Repair Patch-mediated Double Strand DNA Break Formation in Human Cells

The effect of phosphorylation by casein kinase 2 on the activity of insulin-like growth factor-binding protein-3.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is known to be secreted as a phosphoprotein, constitutively phosphorylated at casein kinase 2 (CK2) sites. To examine the effect of phosphorylation by CK2 on the properties of glycosylated human IGFBP-3, we phosphorylated plasma-derived IGFBP-3, containing less than 1 mol/mol phosphoserine, in vitro. As judged by incorporated 32P, enzymatic deglycosylation did not decrease the phosphate content of phospho-IGFBP-3. Phosphorylation had no effect on IGF-I or IGF-II binding, but was inhibitory to acid-labile subunit binding in the presence of either IGF. Determined in simian virus 40-transformed human fibroblasts, cell association by phospho-IGFBP-3 was inhibited approximately 50% compared with that of the nonphosphorylated preparation. Phospho-IGFBP-3 showed significant resistance to proteolysis by plasmin and a cysteine protease secreted by MCF-7 cells. However, no difference was seen between the two preparations in their inhibition of IGF-I-stimulated DNA synthesis when coincubated with IGF-I in neonatal skin fibroblasts or MCF-7 breast cancer cells, and little difference was found in their ability to potentiate IGF-I-stimulated DNA synthesis when preincubated with fibroblasts. These results indicate that IGFBP-3 interaction with acid-labile subunit and with the cell surface, both of which involve basic carboxyl-terminal residues, may be modulated by phosphorylation. Relative resistance to proteolysis and poor binding to cells suggest that CK2-phospho-IGFBP-3 may be a significant inhibitor of IGF activity in the extracellular environment.

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts.

Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10-20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy.

ALTERATION OF THE INSULIN-LIKE GROWTH FACTOR SYSTEM OF MITOGENS IN HYPERPLASTIC BLADDERS OF PARAPLEGIC RATS

Smooth muscle cells of the bladder retain the ability to proliferate in response to injury or mechanical stimulation. Insulin-like growth factor (IGF) I is a mitogenic and hypertrophic agent for smooth muscle cells. The purpose of this study is to examine if spinal cord injury could lead to bladder hypertrophy via the IGF system. The study involved spinal cord transection of female Sprague Dawley rats (approximately 250 to 300 gm.). Six weeks following surgery the urinary bladder was collected. Northern and Western blotting, and IGF-I receptor (IGF-IR) affinity labeling were used to determine the expression of the IGF system, IGFBP levels, and IGF-IR levels respectively. Chronic spinal cord injury leads to an increase in the wet weight of the bladder and in the level of proliferation cell nuclear antigen expression. IGF-I mRNA levels increase, while IGFBP 3 and 5 mRNA and protein levels dramatically decrease. The gene expression of IGFBP 2 and 4 varies from rat to rat, and IGF-IR expression slightly increases. Our results suggest that following spinal cord injury, overexpression of IGF-I and underexpression of IGFBP 3 and 5 may lead to hyperplasia of the smooth muscle layer of the bladder.

Changes of Nuclear Glycoproteins in Normal and Simian Virus 40-Transformed WI-38 Human Lung Fibroblasts

Changes of Nuclear Glycoproteins in Normal and Simian Virus 40-Transformed WI-38 Human Lung Fibroblasts

The 105-kDa Basement Membrane Autoantigen p105 Is N-Terminally Homologous to a Tumor-Associated Antigen

Certain constitutive skin basement membrane components, such as bullous pemphigoid antigens and epidermolysis bullosa acquisita antigen, were discovered because they were targeted by an autoimmune reaction. We aimed to purify and characterize a 105-kDa skin basement membrane protein termed p105 recognized by autoantibodies (anti-p105) from patients with a unique immune-mediated subepidermal blistering skin disease. A simian virus 40-transformed human fibroblast cell line that synthesizes and secretes p105 was utilized as the protein source. p105 was partially purified by salt-gradient fractionation of serum-free conditioned medium through a Mono Q anion-exchange column and by examining each fraction with protein staining and immunoblotting against anti-p105. p105 was isolated from polyacrylamide gel electrophoresis gels, blotted onto polyvinylidene difluoride membrane, and subjected to protein microsequencing. The 20 microsequenced N-terminal amino acids exhibited no homology to known basement membrane proteins but exhibited a 70% homology to a 90-kDa tumor-associated antigen. Antibodies raised against a peptide generated from these amino acid sequences reacted to a 105-kDa western-blotted keratinocyte and fibroblast protein and a basement membrane component. p105 resisted digestion by glycosidases chondroitinase ABC, neuraminidase, and N-glycosidase F but was cleaved by protease V8 to antigenic fragments of 22 kDa and 14 kDa. The synthesis of p105 was inhibited by cycloheximide. We conclude that p105 is a unique basement membrane component produced by both keratinocytes and fibroblasts.

Diminished Expression of Insulin-like Growth Factor (IGF) Binding Protein-5 and Activation of IGF-I-mediated Autocrine Growth in Simian Virus 40-transformed Human Fibroblasts

The reduced growth factor requirements of murine fibroblasts transformed by simian virus 40 (SV 40) have been attributed to insulin-like growth factor (IGF)-I induction by T antigen and consequent activation of IGF-I receptor signaling. The present study shows that the autonomous growth of SV 40-transformed human fibroblasts also requires type-I IGF-I receptor activation but that this is not due to de novo induction of IGF-I gene expression since untransformed human fibroblasts, which fail to proliferate in the absence of serum, also showed IGF-I gene expression under serum-free conditions. DNA synthesis assays confirmed that untransformed cells were responsive to exogenous IGF and indicated that transformed cells were already maximally stimulated. In untransformed fibroblasts, IGF binding was principally to abundant membrane-associated IG-FBP-5, whereas in transformed fibroblasts this protein was minimally expressed, and IGF binding was to IGF receptors. Loss of detectable membrane-associated IG-FBP-5 in transformed cells was associated with diminished IGFBP-5 gene expression and with loss of IGF-II gene expression. Exogenous IGFBP-5 associated with the membranes of transformed cells and inhibited the autocrine growth of these cells. These findings suggest that loss of IGFBP-5 in SV 40-transformed fibroblasts facilitates interaction of endogenously produced IGF-I with the IGF-I receptor and increases their sensitivity to autocrine stimulation.

Senescence of immortal human fibroblasts by the introduction of normal human chromosome 6.

In these studies we show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The loss of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by the analysis for DNA sequences specific for the donor chromosome. Our results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results we propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.

Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies.

Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4 degrees C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.

A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts.

Insulin-like growth factor I and II (IGF-I and IGF-II) associate with specific IGF binding proteins (IGFBPs) present in plasma and extracellular fluids that can modulate the anabolic effects of these peptides. IGF-I has been shown to increase IGFBP concentrations in vivo and in vitro, but the mechanism and significance of this action are unknown. We examined these issues using normal and simian virus 40-transformed adult human fibroblasts (SV40-HF) in culture. Treatment with IGF-I markedly stimulated the appearance of IGFBP-3 (42/38 kD doublet), a 36 kD IGFBP, and 28-32 kD IGFBPs in the medium of these cells, as assessed by Western ligand blotting; IGF-I decreased levels of 24 kD IGFBP in normal HF cultures. The IGF-I-induced change in IGFBP levels was not a type I IGF receptor-mediated effect on IGFBP synthesis because (a) high concentrations of insulin did not mimic IGF-I's effect; (b) IGF-II and IGF-I analogues having reduced affinity for the IGF-I receptor were equipotent with IGF-I in increasing medium IGFBPs; (c) [QAYL]IGF-I, and IGF-I analogue having normal receptor affinity and decreased affinity for IGFBPs, had no effect; and (d) alpha IR-3, a monoclonal antibody specific for the type I IGF receptor, did not block IGF-I-stimulated increases in IGFBPs. In physiological studies, preincubation with 1 nM IGF-I had no effect on type I IGF receptor binding in normal HF and SV40-HF. In contrast, preincubation of cells with an equivalent concentration of [QAYL]IGF-I downregulated the receptors 40-50%. Changes in cell surface receptor number were reflected in cell responsiveness to IGF-I-stimulated [3H]thymidine incorporation and [3H]aminoisobutyric acid uptake. In conclusion, IGF-I regulates the availability of specific IGFBPs in cultured human fibroblasts by a novel receptor-independent mechanism. Rapid changes in levels of soluble IGFBPs as a direct response to extracellular IGF-I, in turn, modulate IGF-I peptide and receptor interaction, and may constitute an important level of control in IGF cellular physiology.

Influence of cellular sequences on instability of plasmid integration sites in human cells

To learn more about mechanisms of genome instability in human cells, I investigated DNA sequences that promote high rates of recombination by analyzing rare unstable plasmid integration sites in simian virus 40-transformed human fibroblasts. Previous studies had hypothesized that rearrangement or loss of integrated sequences could be attributed to adjacent cellular DNA. Consistent with this interpretation, a cloned fragment containing both the integrated plasmid and 2.0 kb of adjacent cell DNA from one such unstable integration site in the cell line LM205 demonstrated a much higher incidence of rearrangements when integrated into other chromosome locations than did the original plasmid. To further test this hypothesis, portions of cellular DNA from this region were integrated in duplicate in other locations to determine their ability to promote restriction-fragment-length polymorphism, an indicator of high rates of homologous recombination. Although two types of instability were observed, neither could be attributed solely to the cell sequences being tested in the plasmid. The first type of instability was a transient deletion or amplification of the plasmid DNA soon after integration, which appeared to be a general phenomenon often associated with any type of newly integrated sequence. A second type of instability continued indefinitely for many cell generations, as did that observed in cell line LM205. Because this was rare (one of 78 clones tested), it could not be attributed solely to cell sequences contained within the plasmid. However, the rearrangements in this cell clone occurred exclusively within the cell DNA adjacent to the integration site, again suggesting a role for cis-acting cell sequences in this process. The inability to identify specific cell sequences responsible for instability may therefore indicate that a complex combination of sequences is involved, possibly within both the plasmid and cell DNA.

Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome.

Anti-Ku (p70/p80) autoantibodies in patients with scleroderma-polymyositis overlap syndrome recognize a 70-kDa/80-kDa protein heterodimer which binds to terminal regions of double-stranded DNA. In the present study, we isolated full-length cDNAs that encode the 80-kDa Ku subunit. Initial screening of a human spleen cDNA library with anti-Ku antibodies yielded a cDNA of 1.0 kilobase (kb) (termed K71) encoding a portion of the 80-kDa Ku polypeptide (identification based on immunological criteria). In RNA blots, this cDNA hybridized with two mRNAs of 3.4 and 2.6 kb. In rescreening of a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA with the K71 cDNA as a hybridization probe, three positive clones were isolated, and that bearing the longest insert (termed Ku80-6) was selected for further characterization. In vitro transcription and translation experiments produced an immunoprecipitable polypeptide which comigrated with the 80-kDa Ku subunit. The Ku80-6 cDNA proved to be 3304 nucleotides in length, with an additional poly(A) tail, closely approximating the size of the larger mRNA. It contains a single long open reading frame encoding 732 amino acids (Mr = 82,713). The putative polypeptide has a high content of acidic amino acids and a region with periodic repeat of leucine in every seventh position which may form the "leucine zipper" structure. In genomic DNA blots, probes derived from the opposite ends of cDNA Ku80-6 hybridized with several nonoverlapping restriction fragments from human leukocyte DNA, indicating that the gene encoding the 80-kDa Ku polypeptide is divided into several exons by intervening sequences.

SV40-transformed normal and DNA-repair-deficient human fibroblasts can be transfected with high frequency but retain only limited amounts of integrated DNA

The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0–40 kb in length. The transfection frequency for the selected (G418 R) marker was between 2 × 10 −4 and 2 × 10 −3 for all cell lines, comparable to many other mammalian systems. About 50% of the G418 R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.

Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation.

Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line. Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated. These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4). We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants. DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences. In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene. In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced. Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations. Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold. The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.

Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation.

Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line. Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated. These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4). We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants. DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences. In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene. In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced. Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations. Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold. The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.

Properties of synthetic-gene recombinant human epidermal growth factor: comparison with the natural growth factor from human urine and milk.

The receptor-binding, immunological and biological properties of synthetic-gene human epidermal growth factor (EGF), produced by recombinant techniques in yeast have been compared with those of mouse submaxillary gland EGF and partially purified EGF from human urine and milk. The three forms of human EGF produced parallel concentration curves in radio-receptor assays using AG2804 Simian virus 40-transformed human lung fibroblasts and iodinated mouse or recombinant human EGF. Equivalent receptor-binding activities of urine and milk EGF were equipotent with recombinant human EGF in an homologous radioimmunoassay using recombinant human EGF antiserum with 125I-labelled recombinant human EGF, while none of the preparations was effective in competing for binding of 125I-labelled mouse EGF to mouse EGF antiserum. Urine, milk and recombinant human EGF stimulated protein synthesis and inhibited protein degradation in cultured AG2804 fibroblasts with identical potency. On a weight basis, mouse EGF was equipotent with recombinant human EGF in competitive binding to cell receptors and in effects on protein synthesis or protein degradation in AG2804 fibroblasts. It is concluded that recombinant human EGF is indistinguishable from the natural growth factor from urine or milk and shares similar biological properties with mouse EGF.