Differentially expressed genes identified in human melanoma cell lines with different metastatic behaviour using high density oligonucleotide arrays.

The increasing incidence of melanoma and the lack of effective treatment, with the exception of tumour excision before the onset of the metastatic phase, make it important to identify genes that may function as new molecular markers for diagnosis and/or prognosis or as new targets for therapy. Recently, a new technique using high density oligonucleotide arrays has been developed to simultaneously screen for the expression of thousands of genes. We used this technique to compare the mRNA expression patterns of two human melanoma cell lines with different metastatic behaviour. Eight differentially expressed genes, namely apolipoprotein CII, tyrosinase-related protein 1, transforming growth factor-beta superfamily, subtilisin-like protein, elongation factor 1 alpha2, alpha2-macroglobulin, human cell division cycle 10 and serine/threonine protein kinase (DYRK1A), were selected to validate the array results by Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, a reliable correlation between differential expression of these genes in the melanoma cell lines and in fresh lesions of melanocytic tumour progression was demonstrated by RT-PCR analysis. Altogether, our data indicate that high density oligonucleotide arrays are a valuable and reliable tool to screen for differentially expressed genes, and that our study may be considered a basic step in the characterization of genes that are involved in the (malignant) progression of melanoma.

ADP-ribosyltransferase from Helix pomatia. Purification and characterization.

ADP-ribosyltransferases from several higher eukaryotes have been purified and characterized, but little is known about ADP-ribosyltransferases in lower eukaryotes. We have purified an ADP-ribosyltransferase (EC 2.4.2.30) from Helix pomatia. The enzyme has an apparent Km of 26.7 microM. Optimal conditions for the enzyme reaction are 17.5 degrees C and pH 8. The time course is linear during the first 10 min of the reaction. The enzyme is capable of poly-ADP-ribosylation. The most highly purified preparation shows one major band at an Mr of 75,000 on electrophoresis in an SDS/polyacrylamide gel, with minor bands at Mr 115,000 and 155,000. Re-activation of SDS/polyacrylamide gels in situ shows the 75,000-Mr band to be enzymically active and additional active bands with Mr values of 115,000, 90,000 and 87,000 respectively. The 115,000-Mr and 75,000-Mr bands cross-react with a polyclonal affinity-purified antiserum against human ADP-ribosyltransferase. Like enzymes from higher eukaryotes, the activity from Helix pomatia is inhibited by thymidine, theophylline, theobromine nicotinamide, 3-methoxybenzamide and 3-aminobenzamide, and is dependent on histone and DNA.

Isolation of a cDNA clone for human NAD+: protein ADP-ribosyltransferase.

NAD+:Protein ADP-ribosyltransferase (EC 2.4.2.30) (ADPRT) was purified from human placenta by affinity chromatography. With the purified enzyme specific antibodies were raised and partial amino acid sequences were determined. To one of the amino acid sequences corresponding oligonucleotides were synthesized. A sized HeLa lambda gt11 cDNA library was constructed and screened. Positive clones were characterized to be ADPRT specific by immuno- and hybridization techniques. Clone ADPRT-G8 reacted with affinity chromatographically purified specific antibodies and with two specific oligonucleotides. The DNA of this clone detected an mRNA of about 4 kb, sufficient in size to code for the ADPRT with an Mr of 116,000. Partial sequence analysis of this clone confirmed its identity by revealing sequences which code for peptides which were found in cyanogen bromide (CNBr) fragments of the purified enzyme. The ADPRT-G8 clone was characterized with respect to its restriction pattern. The cloned ADPRT cDNA now opens the possibility to investigate the role of this enzyme in control of cellular functions.

ADP-ribosyltransferase is highly conserved: purification and characterization of ADP-ribosyltransferase from a fish and its comparison with the human enzyme.

Covalent modification of proteins by ADP-ribosylation is a major mode of protein regulation in eukaryotic cells. ADP-ribosyltransferases have been characterized from mammals but little is known about these enzymes in lower vertebrates. We purified an ADP-ribosyltransferase (E.C. 2.4.2.30) from trout (Salmo trutta faris) by affinity chromatography and characterized it. The 11,700-fold purified activity shows a major protein band at a molecular mass of 75,000 kDa in a SDS-polyacrylamide gel. In situ reactivation of SDS gels showed the 75,000 kDa protein to be enzymatically active, and additional enzymatically active bands at molecular masses of 115,000, 90,000 and 87,000 kDa, respectively. The enzyme is capable of poly-ADP-ribosylation. It crossreacts with affinity purified antibodies raised against human poly(ADP-ribose)synthetase and, except for the temperature optimum, its properties strongly resemble the mammalian enzymes, indicating the conserved character of nuclear ADP-ribosyltransferases. The trout enzyme is DNA- and histone-dependent, has an optimal pH between 8 and 9 and an apparent Km for NAD+ of 24 microM. The temperature optimum is 10 degrees C compared with 25 degrees C for the human enzyme. Known ADP-ribosyltransferase inhibitors also inhibit the enzyme from trout.

Isolation of ADP-ribosyltransferase by affinity chromatography.

An affinity adsorbent for ADP-ribosyltransferase (EC 2.4.2.30) has been synthesized by coupling 3-aminobenzamide to Sepharose 4B. Using this material, ADP-ribosyltransferase from human placenta has been purified from crude extract to homogeneity within a few hours. The enzyme has an apparent Km for NAD+ of 52 microM. Its molecular mass is 115,000 as determined by gel electrophoresis. The enzyme is DNA dependent and stimulated by histone, its temperature optimum is at 25 degrees C, and its pH optimum is around pH 9. alpha-NAD+, thymidine, caffeine, theophylline, theobromine, 3-methoxybenzamide, and nicotinamide inhibit the enzyme. Purification of ADP-ribosyltransferases from horse, rat, and chicken liver was also achieved with the method described.

Transient expression of a plasmid gene, a tool to study DNA repair in human cells: defect of DNA repair in Cockayne syndrome; one thymine cyclobutane dimer is sufficient to block transcription.

Transfected recombinant DNA with regulatory elements such as eukaryotic promoter and termination sites is transiently expressed in human fibroblast cells. Utilizing an expression vector containing the simian virus 40 (SV 40) early control region followed by the E. coli chloramphenicol acetyltransferase (CAT) gene, we investigated the ability of normal, Xeroderma pigmentosum and Cockayne Syndrome cells to repair UV lesions in transfected DNA. Fibroblasts from Xeroderma pigmentosum patients which cannot excise pyrimidine cyclobutane dimers were unable to restore expression of UV irradiated CAT gene. An UV dose inducing one thymine cyclobutane dimer in the transcribed strand of the CAT gene blocked its transcription in these repair deficient cells. Normal cell were able to repair the lesions in transfected DNA during an incubation period of about 40 h and in this way could overcome the UV block. In several fibroblast cell lines from patients suffering from Cockayne Syndrome expression of UV damaged CAT gene was restored significantly less than in normal fibroblasts, indicating that Cockayne Syndrome is associated with a UV repair defect.

Fibroblasts from patients with Fanconi's anemia are not deficient in excision of thymine dimer.

Fibroblasts from a patient with Fanconi's anemia were reported to show a defective excision of pyrimidine dimer [15]. We developed a sensitive radioimmuno assay which is specific for thymine dimer, the main ultraviolet photoproduct, and reinvestigated the thymine dimer excision in fibroblasts from patients with Fanconi's anemia. The analysis of 7 Fanconi's anemia cell lines did not agree with the claim mentioned above that was derived from only one Fanconi's anemia cell line. All cell lines we studied, including the cell line used previously [15], excised thymine dimer from their DNA with excision rates similar to those of normal fibroblasts. Additionally, in two Fanconi's anemia and in two normal fibroblast cell lines the repair capacity was examined.

A synthetic hapten for induction of thymine-dimer-specific antibodies.

High specificity and sensitivity of thymine cyclobutane dimer (thy[]thy) detection were obtained by a radioimmunoassay. Attempts to raise thy[]thy-monospecific antibodies with antigens produced according to conventional methods were unsuccessful. Thy[]thy-specific antibodies could only be raised by using a new strategy to bind thy[]thy to protein: thymine was activated by trimethylsilylation and alkylated at N1 yielding N1-thyminebutanoic acid which was dimerised by ultraviolet treatment. The resulting derivative of thymine cyclobutane dimer was coupled to bovine serum albumin by the active-ester method. The new strategy appears to be generally applicable for binding haptens, such as DNA bases, photoproducts etc, to proteins via a derivative containing a carboxyl group. Immunisation of rabbits with the thy[]thy-bovine-serum-albumin conjugate prepared by the new method resulted in a highly specific antiserum which allows detection of thy[]thy down to 0.06 p mol (15pg). The thy[]thy-specific radioimmunoassay was applied to measure thy[]thy formed in human fibroblasts which were exposed to sunlight at altitudes of 600 m or 2300 m. The amounts of thy[]thy formed in an hour corresponded to doses of 14 J m-2 and 24 J m-2, respectively, of an ultraviolet light lamp emitting predominantly 245-nm light.

DNA repair dependent NAD+ metabolism is impaired in cells from patients with Fanconi's anemia.

In vitro cultivated fibroblasts derived either from patients with Fanconi's anemia (FA) or from healthy probands were analyzed for their DNA repair-dependent NAD+ metabolism. No difference in NAD+ pools was found. NAD+ consumption after cell damage by u.v. irradiation was, however, significantly reduced in FA cells. Several FA cell lines had a lowered ability to transfer ADP-ribose to acid-precipitable material. Additionally, a decreased activity of NAD: protein ADP-ribosyltransferase was found for three FA cell lines. Our data indicate, that FA is accompanied by a defective NAD+ metabolism during DNA repair.

Repair rate in human fibroblasts measured by thymine dimer excorporation.

The UV photoproduct, thymine dimer (-TT), is excorporated with a remarkably low rate from the DNA of human fibroblasts grown in cell culture. An UV dose of 18 J/m2 creates 0.045% -TT (related to thymine). Within the first two days of repair logarithmically growing and quiescent fibroblasts exhibit the same repair rates; thereafter, the proportion of -TT/T is lower in growing cells due to recovery of DNA replication. Only about 50% of the lesions are excised within 24 h. In quiescent cells, 13% of the thymine dimers originally present can be detected as late as a week after UV-irradiation. Two distinct first-order rate constants indicate that approximately half of the dimers are less accessible to repair. Repair measured by the nucleoid decondensation technique corresponds to the faster repair rate, whereas the slow repair rate cannot be detected by this method. Saturation of repair is found beyond 27 J/m2. The remarkably slow rate of excision indicates that thymine dimers are not lethal lesions in human fibroblasts.

A sensitive radioimmuno assay for thymine dimers.

A sensitive radioimmuno assay (RIA) method for detection of the UV photoproduct, thymine dimers (TT) has been developed. The limit of detection of this method is 6 X 10(-14) mol or 15 pg thymine dimer. It is highly specific: A structurally similar compound such as uridine dimer (UU) interferes with the detection of thymine dimers only when it is 53,000-fold or more in molar excess. Since this RIA method does not require the use of labeled DNA, it represents a considerable improvement for repair studies with radiation-sensitive cells.