Dan Canaani

Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG.

Autophagy, the degradation of cytoplasmic components, is an evolutionarily conserved homeostatic process involved in environmental adaptation, lifespan determination and tumour development. The tumor suppressor Beclin1 is part of the PI(3) kinase class III (PI(3)KC3) lipid-kinase complex that induces autophagy. The autophagic activity of the Beclin1–PI(3)KC3 complex, however, is suppressed by Bcl-2. Here, we report the identification of a novel coiled–coil UV irradiation resistance-associated gene (UVRAG) as a positive regulator of the Beclin1–PI(3)KC3 complex. UVRAG, a tumour suppressor candidate that is monoallelically mutated at high frequency in human colon cancers, associates with the Beclin1–Bcl-2–PI(3)KC3 multiprotein complex, where UVRAG and Beclin1 interdependently induce autophagy. UVRAG-mediated activation of the Beclin1–PI(3)KC3 complex promotes autophagy and also suppresses the proliferation and tumorigenicity of human colon cancer cells. These results identify UVRAG as an essential component of the Beclin1–PI(3)KC3 lipid kinase complex that is an important signalling checkpoint for autophagy and tumour-cell growth.

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 transformants 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 cells response to DNA damage is modulated by CKII-dependent protein phosphorylation.

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.

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.

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.

Decoding pooled RNAi screens by means of barcode tiling arrays

BackgroundRNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. In the past years, several pooled large-scale shRNA screens have identified a variety of genes involved in cancer cell proliferation. All of those studies employed microarray analysis, utilizing either the shRNAs half hairpin sequence or an additional shRNA-associated 60 nt barcode sequence as a molecular tag. Here we describe a novel method to decode pooled RNAi screens, namely barcode tiling array analysis, and demonstrate how this approach can be used to precisely quantify the abundance of individual shRNAs from a pool.ResultsWe synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNAs half hairpin sequences. We further demonstrate how barcode tiling arrays can be used to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. Out of a pool of 305 shRNAs, we identified 28 candidate shRNAs to fully or partially impair the viability of the breast carcinoma cell line MDA-MB-231. Individual validation of a subset of eleven shRNA expression constructs with potential inhibitory, as well as non-inhibitory, effects on the cell line proliferation provides further evidence for the accuracy of the barcode tiling approach.ConclusionsIn summary, we present an improved method for the rapid, quantitative and statistically robust analysis of pooled RNAi screens. Our experimental approach, coupled with commercially available lentiviral vector shRNA libraries, has the potential to greatly facilitate the discovery of putative targets for cancer therapy as well as sensitizers of drug toxicity.

Application of the concept synthetic lethality toward anticancer therapy: A promise fulfilled?

Back in 1997, a suggestion to apply the concept of synthetic lethality towards the development of selective, less toxic, cancer drugs and anticancer targets, was brought forward. The availability of large scale synthetic, low-molecular-weight chemical libraries seemed to lend itself to the concept. Human/mouse genome-wide siRNAs and shRNA-expressing libraries allowing high throughput screening for target genes synergistic lethal with defined human cancer aberrations, also raised high hopes of a soon to be established selective therapy. Sixteen years later, the major experimental aspects relating to the implementation of this more focused approach to cancer drug discovery, is briefly and critically reviewed.

shRNA‐mediated RNA interference as a tool for genetic synthetic lethality screening in mouse embryo fibroblasts

Previously, we demonstrated the establishment of synthetic lethality screening in cultured somatic human cells, or mouse embryo fibroblasts (MEFs), for chemicals or mutant genes synergistically lethal with a mutated gene of interest. Here, we show in MEFs that the usage of RNA interference‐based genetic suppressor elements encoding short hairpin RNAs (shRNAs) enables for genetic synthetic lethality screening at a frequency much higher than that achieved before with short truncated sense and antisense RNAs. These findings open up the possibility of using in mammalian cells genome‐wide shRNA libraries for genetic synthetic lethality screening at the multi‐gene level.

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.