Methodological approaches in application of synthetic lethality screening towards anticancer therapy.

A promising direction in the development of selective less toxic cancer drugs is the usage of synthetic lethality concept. The availability of large-scale synthetic low-molecular-weight chemical libraries has allowed HTS for compounds synergistic lethal with defined human cancer aberrations in activated oncogenes or tumour suppressor genes. The search for synthetic lethal chemicals in human/mouse tumour cells is greatly aided by a prior knowledge of relevant signalling and DNA repair pathways, allowing for educated guesses on the preferred potential therapeutic targets. The recent generation of human/rodents genome-wide siRNAs, and shRNA-expressing libraries, should further advance this more focused approach to cancer drug discovery.

Functional conservation between the human, nematode, and yeast CK2 cell cycle genes.

Protein kinase CK2 (formerly casein kinase II) is a highly conserved serine/threonine protein kinase ubiquitous in eukaryotic organisms. Previously, we have shown that CK2 is required for cell cycle progression and essential for the viability of the yeast Saccharomyces cerevisiae. We now report that either the human or the nematode Caenorhabditis elegans CK2alpha catalytic subunit can substitute for the yeast catalytic subunits. Additionally, expression of the human CK2 regulatory subunit (CK2beta) can suppress the temperature sensitivity of either of the two yeast CK2 mutant catalytic subunits. Taken together, these observations reinforce the view that the CK2 cell cycle progression genes have been highly conserved during evolution from yeast to humans, not only in structure but also in function.

Genetic synthetic lethality screen at the single gene level in cultured human cells.

Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.

Establishment of a chemical synthetic lethality screen in cultured human cells.

The synthetic lethality screen is a powerful genetic method for unraveling functional interactions between proteins in yeast. Here we demonstrate the feasibility of a chemical synthetic lethality screen in cultured human cells, based in part on the concept of the yeast method. The technology employs both an immortalized human cell line, deficient in a gene of interest, which is complemented by an episomal survival plasmid expressing the gene of interest, and the use of a novel double-label fluorescence system. Selective pressure imposed by any one of several synthetic lethal metabolic inhibitors prevented the spontaneous loss of the episomal survival plasmid. Retention or loss over time of this plasmid could be sensitively detected in a blind test, while cells were grown in microtiter plates. Application of this method should thus permit high throughput screening of drugs, which are synthetically lethal with any mutant human gene of interest, whose normal counterpart can be expressed. This usage is particularly attractive for the search of drugs, which kill malignant cells in a gene-specific manner, based on their predetermined cellular genotype. Moreover, by replacing the chemicals used in this example with a library of either DNA oligonucleotides or expressible dominant negative genetic elements, one should be able to identify synthetic lethal human genes.

Molecular cloning of a novel human gene encoding a 63-kDa protein and its sublocalization within the 11q13 locus.

A human cDNA previously isolated by virtue of its ability to complement partially the ultraviolet sensitivity of a xeroderma pigmentosum cell line was further characterized. The transcription unit is expressed as a single 4.0-kb mRNA that encodes a novel 63-kDa cytoplasmic protein, possibly initiating from an internal AUG codon. The gene encoding this protein, named UVRAG, has been extremely well conserved during evolution, implying an important role for this gene product in cell metabolism. The transcribed mRNA is constitutively expressed in a wide variety of human tissues. The protein encoded by this gene is predicted to contain a coiled-coil structure and is likely to be metabolically unstable based on the occurrence of a strong PEST domain. UVRAG was assigned to human chromosome 11 by Southern hybridization to a somatic cell hybrid panel. Fluorescence in situ hybridization coupled with PCR analysis of human/rodent somatic cell hybrids containing segments of human chromosome 11 has localized this gene to a subregion of 11q13 in between the D11S916 and the D11S906 loci. Importantly, this region has been shown to be amplified in a variety of human malignancies, including breast cancer.

Mapping of the human casein kinase II catalytic subunit genes: two loci carrying the homologous sequences for the alpha subunit.

The human serine/threonine protein casein kinase II (CK II) contains two distinct catalytic subunits, alpha and alpha 1, which are encoded by different genes. A combination of segregation analysis of rodent-human hybrid cells and chromosomal in situ hybridization have localized the human CK II-alpha DNA sequence to two loci: 11p15.5-p15.4 and 20p13. In contrast, the CK II-alpha' gene has been mapped to chromosome 16 by somatic cell hybrid analysis. Taken together with our previous assignment of the CK II regulatory beta-subunit gene to 6p12-p21, these results indicate that although the products of these genes form a single biological complex, they are encoded on different human chromosomes. Further analysis should determine whether both loci of CK II-alpha are functional, or perhaps one of the two constitutes a pseudogene.

Expression of the cDNA for the beta subunit of human casein kinase II confers partial UV resistance on xeroderma pigmentosum cells.

An immortalized xeroderma pigmentosum cell line belonging to the complementation group D (XP-D) was transfected with a normal human cDNA clone library constructed in a mammalian expression vector. Following UV-irradiation-selection, a transformant having a stable, partially UV-resistant phenotype was isolated. A transfected cDNA of partial length was rescued from the transformant's cellular DNA by in vitro amplification, using expression-vector specific oligonucleotides as primers in a polymerase chain reaction (PCR). Expression of this cDNA complemented the UV sensitivity of the XP-D cell line to the UV-resistance levels characteristic of the primary transformant. The nucleotide sequence of the cDNA was determined. The deduced protein identified the cDNA as encoding for the beta subunit of casein kinase II (CKII-beta). Similar to the effect exerted by the truncated CKII-beta cDNA, expression of a cDNA clone encompassing the complete translated region of CKII-beta leads to XP-D cells partially resistant to UV-irradiation. However, transfection of CKII-beta cDNA could also partially complement the UV-sensitivity of a xeroderma pigmentosum cell line belonging to group C (XP-C). Analysis by Southern, Northern and RNAase mismatch cleavage techniques did not reveal any functional defect in the CKII-beta gene of cell lines derived from either 7 XP-D or 10 XP-C families. We therefore consider it unlikely that either the XP-D or the XP-C DNA repair deficiency is associated with a defect in the beta subunit of casein kinase II. Nevertheless, our findings suggest the possibility that the cell's response to DNA damage is modulated by CKII-dependent protein phosphorylation.

Isolation by polymerase chain reaction of a cDNA whose product partially complements the ultraviolet sensitivity of xeroderma pigmentosum group C cells.

A xeroderma pigmentosum (XP) cell line from complementation group C has been complemented to attain ultraviolet (UV) resistance and DNA repair proficiency, by transfection with a human expression cDNA library, followed by selection to UV resistance. We now show that the transfected cDNAs can be rescued from cellular DNA of a secondary transformant by its in vitro amplification using expression-vector-specific oligodeoxyribonucleotides as primers in a polymerase chain reaction. The amplified cDNAs were cloned into a mammalian expression vector. Their transfection into XP cells identified a single cDNA which specifically complemented the UV sensitivity of a group-C-derived cell line to the same partial UV-resistance levels exhibited by the transformant from which the cDNAs were rescued.

Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient.

In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced [3H]thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line.

A hypodiploid karyotype found in immortal human cells selected from a wide spectrum of posttransformation chromosomal complements.

A simian virus 40 (SV40) DNA fragment, encompassing the whole early region but having a defective origin of DNA replication, was previously used to transform human fibroblast cells derived from a patient suffering from xeroderma pigmentosum complementation group C (XP-C). Two independent SV40 transformants had acquired immortality in culture. Unlike most SV40-transformed human fibroblasts, the two established XP-C cell lines possessed an identical hypodiploid karyotype of 44,XX,-19,Xq+,-22,15p+. We now show that prior to immortalization the two SV40 transformants display a very wide spectrum of karyotypes with regard to chromosome number. A similar variety of chromosomal complements is present in four independent mortal SV40 transformants of the same parental XP-C cell line as well as in a mortal SV40-transformed xeroderma pigmentosum group D cell line. The rarity of the immortalization event, coupled with the coincident occurrence of identical karyotypes in the two immortal cell lines, suggests that the immortal lines arose through selection of a peculiar karyotype from among those of the parent SV40-transformed fibroblasts, and that this peculiar hypodiploid karyotype may be related to, and perhaps even necessary for, the establishment of immortality.

Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library.

In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion.

Immortalization of xeroderma pigmentosum cells by simian virus 40 DNA having a defective origin of DNA replication.

A simian virus 40 (SV40) DNA fragment, encompassing the whole early region and having a defective origin of DNA replication, has been used to transform human fibroblast cells derived from two xeroderma pigmentosum (XP) patients. Two of the SV40-transformed XP cell lines, belonging to complementation group C, had acquired the characteristic of indefinite life-span in culture. These XP cell lines synthesize T antigen as shown by immunofluorescence and retain the high sensitivity to UV irradiation. Detailed karyotype analysis shows very few chromosomal changes, while the transfecting SV40 DNA is integrated into cellular DNA sequences. These are the first immortalized XP cell lines derived from complementation group C. In view of the extreme difficulty in obtaining immortalized human fibroblasts, we suggest a possible advantage of replication defective SV40 DNA molecules for immortalizing human fibroblast cells of any source.

Regulated expression of human interferon beta 1 gene after transduction into cultured mouse and rabbit cells.

The human interferon beta 1 gene has been inserted into simian virus 40 hybrid plasmid vectors carrying the bacterial phosphotransferase gene (neo), and introduced into cultured mammalian cells by DNA transfection. A majority of the transformants resistant to the antibiotic G418 were capable of synthesizing and secreting biologically active human interferon. The neo/interferon transformants contain several copies of the transfecting DNA integrated into cellular DNA sequences. In most transformants the production of human interferon and its mRNA is induced by the addition of poly(rI) X poly(rC); by contrast, the level of neo mRNA is not increased under the same conditions. The 5' end of the human interferon mRNA produced after induction was indistinguishable from the interferon mRNA induced in human fibroblasts. This indicates that information enabling human beta 1 interferon gene to be induced by poly(rI) X poly(rC) is localized to sequences within, or 5'-proximal to, the coding sequence.

Specific in vitro initiation of transcription of simian virus 40 early and late genes occurs at the various cap nucleotides including cytidine.

High specific activity [beta-32P]ATP and [beta-32P]CTP were used to study in vitro transcriptional initiation and subsequent capping of simian virus 40 (SV40) early and later RNAs. More than 40% of the capped SV40 RNA synthesized in vitro was also polyadenylylated. With [beta-32P]ATP, only adenosine-containing caps were labeled and the incorporated radioactive phosphate was found exclusively in the beta position. Cap digestion patterns showed extensive qualitative and quantitative similarities between these 32P-labeled caps and caps labeled in vivo [Canaani, D., Kahana, C., Mukamel, A. & Groner, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 3078--3082]. With [beta-32P]CTP, only early SV40 RNA was labeled, consistent with the absence of cytosine-containing caps in late transcripts. The [beta-32P]CTP-labeled cap was identified as m7GpppCmpU, which was previously identified as the major cap of in vivo labeled early SV40 mRNA [Kahana, C., Gidoni, D., Canaani, D. & Groner, Y. (1981) J. Virol. 37, 7--16]. This experiment provides biochemical evidence for eukaryotic RNA polymerase II initiation of transcription with CTP. The data imply that, on SV40 DNA, RNA polymerase II initiates transcription at multiple nucleotide sequences and capping occurs at the initiator nucleotide.

Simian virus 40 early mRNA's in lytically infected and transformed cells contain six 5'-terminal caps.

Late simian virus 40 (SV40) mRNA contains eight different cap structures which we have previously identified and mapped on the viral genome. As reported here, 5'-cap heterogeneity is a common feature to both the early and the late SV40 mRNA's. methyl-3H-labeled viral mRNA was purified from cells infected at 41 degrees C with SV40 mutant tsA209. Three different cap cores were identified: m7GpppGm, m7GpppCm, and m7GpppAm. An average of three to four m6A residues per mRNA molecule was found. RNase T2-resistant 32P-labeled early caps from tsA209-infected cells isolated and characterized. Six distinct cap I structures were identified: m7GpppCmpU (30%), m7GpppGmpC (24%), m7GpppAmpG (18%), m7GpppGmpU (13%), m7GpppGmpG (12%), and m7GpppAmpU (3%). A similar 5'-end heterogeneity was observed in early SV40 mRNA from BSC-1 cells infected with wild-type SV40 strain 777 in the presence of cytosine arabinoside and in the SV40 UV-transformed permissive line C-6. Five of these capped dinucleotides are complementary to DNA sequences at 0.66 map unit in a region previously identified by the primer extension method (Reddy et al., J. Virol. 30:279-296, 1979; Thompson et al., J. Virol. 31:437-438, 1979) as the 5' end of the early message. DNA sequences upstream from this region contain the TATTTAT (Hogness-Goldberg box), which is missing from upstream of the 5'-cap sites of late SV40 mRNA. Thus, 5'-end heterogeneity is not necessarily related to the presence or the absence of this putative transcriptional "initiation signal." When the possibility that SV40 5' caps represent transcriptional initiation sites is considered, the data also suggest that, on SV40 DNA, eucaryotic RNA polymerase II initiates transcription at multiple nucleotide sequences, including pyrimidines.

Sequence heterogeneity at the 5' termini of late simian virus 40 19S and 16S mRNAs.

The 5'-cap-containing leader sequence of the most abundant 19S and 16S mRNAs of simian virus 40 (SV40) was previously mapped between 0.67 and 0.76 map units. We now find that the two late mRNA species contain multiple 5' ends. Eight different RNase T2-resistant cap structures were identified:m7GpppmAmpU (47%); m7GpppmAmpUmpU (19%); m7GpppmAmpC (16%); m7GpppmAmpCmpA (5%); m7GpppmAmpG (6%); m7GpppGmpC (3%); m7GpppmAmGmpA (2%); m7GpppGmpCmpG (2%). Capped T1 oligonucleotides of 19S and 16S mRNAs have been isolated by two different procedures: (i) chromatography on a DEAE-cellulose column followed by paper electrophoresis and (ii) two-dimensional electrophoresis/homochromatography. Cap structures of the isolated 5' oligonucleotides were identified. Each of the major caps was found to be associated with a few differential 5' oligonucleotides, implying a vast heterogeneity at the termini of SV40 late mRNAs. The results suggest that on SV40 DNA, RNA polymerase II has a reportoire of initiation points. In most of the cases, initiation takes place with adenosine triphosphate followed by a pyrimidine. Alternatively, transcription may start at one specific point but a unique mechanism of processing generates heterogeneous populations of termini with a common 5' adenosine triphosphate.