In-gel Renaturation Research Articles

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes. In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Identification of a Broad-Spectrum Peptidoglycan Hydrolase Associated with the Particle of Xanthomonas oryzae Phage Xop411

Virion-associated peptidoglycan hydrolases (VAPGH) in bacteriophages are potential antimicrobials. Xop411 is a syphophage infecting the Gram-negative Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight in rice plants. The Xop411 gp21 protein was identified here as a peptidoglycan glycohydrolase by Western blotting and zymogram assay, and localized to the phage tail by immunogold-labelling electron microscopy. This protein showed an apparent molecular mass of 17 kDa in SDS-polyacrylamide gels, larger than that calculated from the amino acid sequence, 15 kDa with 130 residues. The recombinant gp21 expressed in Escherichia coli formed inclusion bodies, which gained enzyme activity after in-gel renaturation. In contrast, the secreted recombinant protein (s-gp21His) expressed in Pichia pastoris was soluble and enzymatically active. Plate assays showed that s-gp21His was capable of killing 3 species of Xanthomonas, a genus containing 27 closely related plant pathogenic species, as well as the opportunistic Pseudomonas aeruginosa and Stenotrophomonas maltophilia causing nosocomial infections. These results indicate that the Xop411 gp21 has possible wide applications as an antimicrobial against xanthomonads and at least 2 opportunistic bacteria. Several other VAPGH from Xanthomonas phages were also identified by bioinformatic analysis, with 1 being confirmed by Western blotting.

A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening

In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases.The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection.

Use of phosphoproteomics to study tyrosine kinase activity in capacitating boar sperm: Kinase activity and capacitation

It is generally accepted that sperm capacitation is associated with the protein kinase A-mediated appearance of tyrosine phosphoproteins, although the substrates and kinase(s) involved have not been identified. We described a Mr 32,000 tyrosine phosphoprotein, “p32”, appearing in porcine sperm coincident with capacitation. We also discovered a tyrosine kinase-like enzyme in boar sperm of Mr 32,000 (“TK-32”) with enhanced activity during capacitation. The present work was conducted to further characterize and to identify these capacitation-related protein(s). Fresh porcine sperm were incubated to induce capacitation then immunoprecipitation, immunoblotting and proteomic analysis revealed seven tyrosine-phosphorylated proteins aligned in the range of Mr 30,000 with different isoelectric pH values (p I). Therefore, p32 may be composed of several tyrosine phosphoproteins. Three were identified as acrosin-binding sp32 (p I 6.5), and two triosephosphate isomerase isoforms (p I 7.1 and 7.9). At present, however, proteonomic analysis has not revealed any kinase at Mr 32,000. Immunoprecipitation experiments show that p32 and TK-32 are different molecules, as TK-32 activity remains in the supernatant of the antiphosphotyrosine precipitates. Finally, in-gel renaturation and immunoblotting suggest that TK-32 is a mitogen-activated protein kinase (MAPK). The discovery of p32 and the MAPK-like TK-32 provides new insight regarding the mechanisms underlying capacitation in the pig.

Purification and identification of a protein kinase activity modulated by ionizing radiation

In order to identify novel protein kinases, which are involved in signal transduction processes after exposure of cells to ionizing radiation we screened HL-60 cells using an in-gel renaturation assay. Using this approach we identified a renaturable serine/threonine kinase with an apparent molecular mass of 90 kDa (pK90). The activity of pK90 dropped within minutes after exposure to a dose of 10 Gy. It reached a minimum 15–30 min after irradiation and increased back to pre-treatment values 6 h later. A down-regulation of the kinase activity was detectable after a dose of 1 Gy. Failure of H 2O 2 to down-regulate pK90 activity indicates a requirement for DNA double-strand-breaks to modulate the kinase activity. In contrast to the molecular mass of 90 kDa in SDS–PAGE we found a molecular mass of around 450 kDa for the native protein using gel filtration chromatography, indicating that pK90 forms a multi-protein complex under native conditions. To identify pK90 we partially purified the protein by three affinity chromatography steps (heparin–Sepharose, phosphate metal affinity, and Cibacron-Blue-F3G-A–Sepharose). Mass spectrometric analysis of the purified 90 kDa fraction showed that pK90 is identical to Tlk1, which was verified by immunoprecipitation.

Casein kinase 1 is tightly associated with paired-helical filaments isolated from Alzheimer's disease brain.

The protein kinase activity tightly associated with paired helical filaments (PHFs) purified from the brain tissue of individuals with Alzheimer's disease has been characterized in vitro. The activity is shown to phosphorylate casein, an exogenous substrate, with a maximal velocity of approximately 2 nmol/min/mg, suggesting it comprises a significant component of the total protein in the PHF preparation. On the basis of substrate selectivity, isoquinoline sulfonamide inhibitor selectivity, in-gel renaturation assays, and western analysis, the activity consists of closely related members of the alpha branch of the casein kinase 1 family of protein kinases. Because of its tight association with PHFs and its phosphate-directed substrate selectivity, casein kinase 1 is positioned to participate in the pathological hyperphosphorylation of tau protein that is observed in neurodegenerative diseases such as Alzheimer's disease.

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens.

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50,900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42 degrees C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 micrograms/ml) and not inhibited by antipain dihydrochloride (120 micrograms/ml), aprotinin (4 micrograms/ml), bestatin (80 micrograms/ml), chymostatin (50 micrograms/ml), E-64 (20 micrograms/ml), leupeptin (4 micrograms/ml), Pefabloc SC (2000 micrograms/ml), pepstatin (4 micrograms/ml), phosphoramidon (660 micrograms/ml), or phenylmethylsulfonyl fluoride (400 micrograms/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.

Activation of Extracellular Signal-Regulated Kinases 1 and 2 (ERK1 and ERK2) by FGF-2 and PDGF-BB in Normal Human Osteoblastic and Bone Marrow Stromal Cells: Differences in Mobility and In-Gel Renaturation of ERK1 in Human, Rat, and Mouse Osteoblastic Cells

We recently demonstrated that basic fibroblast growth factor (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) mainly activated extracellular signal-regulated kinase 2 (ERK2) in normal human osteoblastic (HOB) and bone marrow stromal (HBMS) cells by an “in-gel” MAP kinase assay, although both ERK1 and ERK2 proteins were present. In the present study, we examined whether ERK1 is also activated by growth factors by using three different MAPK assay procedures, an “in-gel MAP kinase assay,” an immune-complex kinase assay, and western blotting with anti-active MAPK antibody which recognizes specifically activated forms of both ERK1 and ERK2. Results have demonstrated that in addition to ERK2, ERK1 is activated by FGF-2 and PDGF-BB in normal HOB and HBMS cells. The human ERK1 moved faster on SDS-polyacrylamide gel compared to rat and mouse, revealing differences in the apparent molecular weight of ERK1 in normal human osteoblastic and bone marrow osteoprogenitor cells, human (TE-85) and rat (ROS 17/2.8 and UMR-106) osteosarcoma, and mouse (MC3T3-E1) osteoblastic cells. ERK1 is less stable in the in-gel renaturation process compared to ERK2; thus, in-gel MAP kinase assay does not provide an accurate estimation of ERK1 activity. Results also showed that anti-active MAPK antibody can be used reliably and accurately to measure the activation of ERK1 and ERK2 in osteoblastic cells.

Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets.

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol esterstimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 microM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST-MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.

Electrophoretic isolation of extrachromosomal DNA from tumor cells.

Gene amplification allows transformed cells to overexpress specific genes and gain a survival advantage. For this reason, cloning and characterization of amplified genes can improve our understanding of the biology of transformed cells. The techniques of in-gel renaturation and chromosome microdissection can enrich for amplified DNA sequences, but both are labor intensive and have other drawbacks. We have developed an alternative strategy of enriching for amplified DNA sequences that involves two-directional agarose gel electrophoresis of extrachromosomal circular DNA. Extrachromosomal circles can be detected with repetitive DNA probes and can be used to produce DNA probes suitable for fluorescence in situ hybridization for location of genomic origin. The ability to enrich for amplified DNA without specialized equipment or transformed cell metaphases should prove useful in the search for new genes which are important in tumor cell progression.

Characterization of 45-kDa/54-kDa HSP27 kinase, a stress-sensitive kinase which may activate the phosphorylation-dependent protective function of mammalian 27-kDa heat-shock protein HSP27.

Heat-shock protein 27 (HSP27) is a major target of phosphorylation upon cell stimulation with a variety of agents and has been suggested to have a phosphorylation-regulated function at the level of actin filaments. Here we investigated comparatively the mechanisms of HSP27 phosphorylation by oxidative stresses, exposures to tumor necrosis factor (TNF), heat shock and growth factors. Extracts of Chinese hamster or human cells exposed to H2O2, xanthine/xanthine oxidase, menadione or TNF contained up to 15-fold more HSP27 kinase activity than comparable extracts obtained from control cells. Induction of HSP27 kinase activity by TNF or H2O2 was completely inhibited by first treating the cells with the antioxidant N-acetyl-L-cysteine, suggesting that generation of reactive oxygen metabolites was the key triggering element of this induction. In contrast, prior treatment with acetylcysteine had no or little effect on the induction by thrombin, serum and heat shock. The kinase activity in extracts of cells stimulated by heat shock, H2O2, sodium arsenite, TNF or growth factors was identified by in-gel renaturation and purified approximately 8000-fold by sequential chromatography. In all cases, the induced kinase activity was entirely associated with two polypeptides of 45 kDa and 54 kDa, identified as mitogen-activated-protein kinase-activated protein (MAPKAP) kinase-2 based on its reactivation in vitro by 42/44-kDa MAP kinases, its antigenic properties and its substrate specificity. The 45/54-kDa HSP27 kinase may play an important role in the cell response to oxidative stress. Overexpression of the wild-type HSP27 but not of a nonphosphorylatable form of human HSP27 in Chinese hamster cells conferred resistance to actin fragmentation by oxidative stress generated by H2O2. It is concluded that activation of the 45/54-kDa HSP27 kinase is a common mechanism of HSP27 phosphorylation to which converge both oxyradical-dependent and oxyradical-independent pathways and which may participate in a homeostatic response to stress at the level of actin microfilament.

Gene amplification in non-Hodgkin's lymphoma

Among 426 consecutively ascertained and karyotypically abnormal non-Hodgkin's lymphoma (NHL) tumours, cytological evidence for gene amplification in the form of homogeneously staining regions (HSRs) was encountered in nine cases of large cell diffuse lymphoma (LC-DL). The mean age of patients with HSRs was 62.9 years and four died within a year of diagnosis. To identify candidate gene(s) amplified in these tumours, we performed a Southern blot analysis of tumour DNA using probes for 23 known protooncogenes and the multidrug resistance gene, PGY1. Besides a two-fold amplification of the BCL2 gene in two cases, no evidence for overt amplification of any of the genes assayed was found. To confirm DNA amplification in these specimens we performed the DNA in-gel renaturation assay. Evidence for presence of amplified DNA fragments was obtained in four of seven specimens. These results suggest amplification of a novel gene(s). To our knowledge, this is the first formal study of gene amplification in a large consecutively ascertained series of fresh lymphoma biopsies.

Novel DNA sequences at chromosome 10q26 are amplified in human gastric carcinoma cell lines: molecular cloning by competitive DNA reassociation

Molecular cloning of genomic sequences altered in cancer cells is believed to lead to the identification of new genes involved in the initiation and progression of the malignant phenotype. DNA amplification is a frequent molecular alteration in tumor cells, and is a mode of proto-oncogene activation. The cytologic manifestation of this phenomenon is the appearance of chromosomal homogeneously staining regions (HSRs) or double minute bodies (DMs). The gastric carcinoma cell line KATO III is characterized by a large HSR on chromosome 11. In-gel renaturation analysis confirmed the amplification of DNA sequences in this cell line, yet none of 42 proto-oncogenes that we tested is amplified in KATO III DNA. We employed the phenol-enhanced reassociation technique (PERT) to isolate 21 random DNA fragments from the amplified domain, and used 6 of them to further clone some 150 kb from that genomic region. While in situ hybridization performed with some of these sequences indicated that in KATO III they are indeed amplified within the HSR on chromosome 11, somatic cell hybrid analysis and in situ hybridization to normal lymphocyte chromosomes showed that they are derived from chromosome 10, band q26. The same sequences were found to be amplified in another gastric carcinoma cell line, SNU-16, which contains DMs, but were not amplified in other 70 cell lines representing a wide variety of human neoplasms. One of these sequences was highly expressed in both KATO III and SNU-16. Thus, the cloned sequences supply a starting point for identification of novel genes which might be involved in the pathogenesis of gastric cancers, and are located in a relatively unexplored domain of the human genome.

Cytogenetic and molecular analysis of human male germ cell tumors: Chromosome 12 abnormalities and gene amplification

We report karyotypic analysis of 24 male germ cell tumors (GCTs) with clonally abnormal karyotypes biopsied from testicular and extragonadal lesions from 20 patients belonging to the histologic categories seminoma, teratoma, embryonal carcinoma, choriocarcinoma, and endodermal sinus tumor. Chromosomes 1, 7, 9, 12, 17, 21, 22, and the X chromosome were nonrandomly gained in these tumors. Nonrandom structural changes affected most frequently chromosomes 1 and 12, the latter as i(12p) and/or del(12)(q13----q22). The i(12p) was seen in 90% of tumors which included all histologic subtypes and gonadal as well as extragonadal presentation. Our present results, along with those from published data on fresh GCT biopsies, establish that i(12p) is a highly nonrandom chromosome marker of all histologic as well as anatomic presentations of GCTs. in contrast, we found del(12)(q13----q22) exclusively in nonseminomatous GCTs (NSGCTs) and mixed GCTs (MGCTs) occurring in 44% of such lesions. Because successful cytogenetic analysis of fresh tumor specimens is not always possible, we developed a method based on DNA analysis to detect i(12p) as increased copy number of 12p. In addition to the changes affecting chromosome 12 identified above, we have detected, for the first time, cytological evidence of gene amplification in the form of homogeneously staining regions (HSRs) and double minute chromosomes (dmins) in treated as well as untreated primary extragonadal and metastatic GCTs and confirmed the presence of amplified DNA in one of these tumors at the molecular level by the in-gel renaturation method. Hybridization of DNA from cultured cells from an HSR-bearing tumor with a panel of probes for genes known to be amplified or otherwise perturbed in diverse tumor systems did not identify the amplified gene, suggesting amplification of a novel gene or genes. This study comprises the largest series of GCT cytogenetics attempted so far. Notably, it includes data on a series of primary mediastinal tumors, a group which previously has not been studied in any detail.

Gene amplification affecting O6-alkylguanine-DNA alkyltransferase activity is not detected in nitrosourea resistant or sensitive human cell lines

An attempt was made to characterize the genetic regulation of the human DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) in the absence of the cloned gene. Four human cell lines, differing in AGT activity from very proficient to essentially absent, were assayed for gene amplification as a possible mediator of the methylation repair phenotype (Mer+, AGT activity and MER-, no AGT activity) using in-gel DNA renaturation and G-banded karyotype analysis. The former technique allows subsequent analysis of amplification units and cloning of observed amplified DNA fragments, a hopeful approach to the isolation of the human AGT gene. Within the sensitivities of the techniques, no correlation between AGT activity and gene amplification was observed in the four cell lines tested.

High-resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation analysis.

Utilizing an in vivo labeling method on synchronized cultures, we have previously defined a 28-kilobase (kb) replication initiation locus in the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) (N. H. Heintz and J. L. Hamlin, Proc. Natl. Acad. Sci. USA 79:4083-4087, 1982; N. H. Heintz and J. L. Hamlin, Biochemistry 22:3552-3557, 1983; N. H. Heintz, J. D. Milbrandt, K. S. Greisen, and J. L. Hamlin, Nature [London] 302:439-441, 1983). To locate the origin of replication in this 243-kb amplicon with more precision, we used an in-gel renaturation procedure (I. Roninson, Nucleic Acids Res. 11:5413-5431, 1983) to examine the labeling pattern of restriction fragments from the amplicon in the early S phase. This method eliminates background labeling from single-copy sequences and allows quantitation of the relative radioactivity in individual fragments. We used this procedure to follow the movement of replication forks through the amplicons, to roughly localize the initiation locus, and to estimate the rate of fork travel. We also used a slight modification of this method (termed hybridization enhancement) to illuminate the labeling pattern of smaller restriction fragments derived solely from the initiation locus itself, thereby increasing resolution. Our preliminary results suggest that there are actually two distinct initiation sites in the amplicon that are separated by approximately 22 kb.

High-resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation analysis.

Utilizing an in vivo labeling method on synchronized cultures, we have previously defined a 28-kilobase (kb) replication initiation locus in the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) (N. H. Heintz and J. L. Hamlin, Proc. Natl. Acad. Sci. USA 79:4083-4087, 1982; N. H. Heintz and J. L. Hamlin, Biochemistry 22:3552-3557, 1983; N. H. Heintz, J. D. Milbrandt, K. S. Greisen, and J. L. Hamlin, Nature [London] 302:439-441, 1983). To locate the origin of replication in this 243-kb amplicon with more precision, we used an in-gel renaturation procedure (I. Roninson, Nucleic Acids Res. 11:5413-5431, 1983) to examine the labeling pattern of restriction fragments from the amplicon in the early S phase. This method eliminates background labeling from single-copy sequences and allows quantitation of the relative radioactivity in individual fragments. We used this procedure to follow the movement of replication forks through the amplicons, to roughly localize the initiation locus, and to estimate the rate of fork travel. We also used a slight modification of this method (termed hybridization enhancement) to illuminate the labeling pattern of smaller restriction fragments derived solely from the initiation locus itself, thereby increasing resolution. Our preliminary results suggest that there are actually two distinct initiation sites in the amplicon that are separated by approximately 22 kb.

The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure.

We have previously cloned and characterized two different dihydrofolate reductase amplicon types from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). The largest of these (the type I amplicon) is 273 kilobases (kb) in length. In the present study, we utilized clones from the type I amplicon as probes to analyze the size and variability of the amplified DNA sequences in five other independently isolated methotrexate-resistant Chinese hamster cell lines. Our data indicated that the predominant amplicon types in all but one of these cell lines are larger than the 273-kb type I sequence. In-gel renaturation experiments as well as hybridization analysis of large SfiI fragments separated by pulse-field gradient gel electrophoresis showed that two highly resistant cell lines (A3 and MK42) have amplified very homogeneous core sequences that are estimated to be at least 583 and 653 kb in length, respectively. Thus, the sizes of the major amplicon types can be different in different drug-resistant Chinese hamster cell lines. However, there appears to be less heterogeneity in size and sequence arrangement within a given methotrexate-resistant Chinese hamster cell line than has been reported for several other examples of DNA sequence amplification in mammalian systems.

Analysis of gene amplification in human tumor cell lines.

Oncogene amplification has been observed in various primary tumors and tumor-derived cell lines. In several types of cancer, amplification of specific oncogenes is correlated with the stage of tumor progression. To estimate the frequency of gene amplification in other tumor types and to determine whether the ability to grow in vivo is associated with gene amplification in tumor cell lines, we have developed a modified version of the in-gel renaturation assay that detects human DNA sequences of unknown nature amplified as little as 7- to 8-fold. This assay was used to screen 16 cell lines derived from various solid tumors and leukemias. Amplified DNA sequences were detected in only one cell line, Calu-3 lung adenocarcinoma. This cell line was found to contain coamplified NGL (formerly termed neu) and ERBA1 oncogenes. However, when one of the amplification-negative cell lines, PC-3 prostatic carcinoma, was selected for in vivo growth in nude mice, amplified DNA sequences became detectable in these cells. The amplified sequences included the MYC oncogene, which showed no amplification in the parental cell line but was amplified 10- to 12-fold in the in vivo-selected cells. MYC amplification may, therefore, provide tumor cells with a selective advantage specific for in vivo growth.