Shoshana Klein

Killing time for cancer cells

As the signalling pathways that control cellular proliferation and death are unravelled, a range of targets have emerged as candidates for molecular cancer therapy. For their survival, cancer cells depend on a few highly activated signalling pathways; inhibition of these pathways has a strong apoptotic effect and can lead to tumour regression. But drugs that exploit this weakness, such as imatinib, have not cured patients: withdrawal of the drug leads to disease recurrence, and sustained treatment leads to the emergence of drug-resistant clones. Can cancer be cured, or will it have to be controlled as a chronic disease?

Targeting the EGFR and the PKB pathway in cancer.

The EGFR and PKB pathways are frequently activated in cancer, so are prime targets for cancer therapy. To this end, new inhibitors are being tested. EGFR inhibitors as single therapy have little benefit, although therapies that evoke an antitumor immune response are more effective. Resistance mutations within the EGFR are common, as is activation of the antiapoptotic PKB pathway via alternative tyrosine kinase receptors, especially other EGFR family members or IGF1R. To combat resistance, multitargeted EGFR inhibitors and combined inhibition of the EGFR and PKB are being investigated. Inhibition of the EGFR and PKB pathways also sensitizes cancer cells to chemotherapy. Thus, EGFR and PI3K/PKB inhibitors will be most effective when used in rational combinations of targeted inhibitors and traditional chemotherapy.

Switching yeast from meiosis to mitosis: double-strand break repair, recombination and synaptonemal complex

When Saccharomyces cerevisiae cells that have begun meiosis are transferred to mitotic growth conditions (‘return‐to‐growth’, RTG), they can complete recombination at high meiotic frequencies, but undergo mitotic cell division and remain diploid. It was not known how meiotic recombination intermediates are repaired following RTG. Using molecular and cytological methods, we investigated whether the usual meiotic apparatus could repair meiotically induced DSBs during RTG, or whether other mechanisms are invoked when the developmental context changes.

G‐Protein Subunit Dissociation Is not an Integral Part of G‐Protein Action

The concept that a guanosine triphosphate (GTP) binding protein (or G protein) is a transducer of receptor-to-effector signal transduction was formulated in the 1970s for hormonedependent adenylyl cyclase. It has been shown that binding of the hormone to the receptor triggers exchange of guanosine diphosphate (GDP) for GTP on the G protein, thereby converting the G protein from the inactive conformation to the activated form. In its GTP-bound state, the G protein activates adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP). Hydrolysis of the GTP terminates the signal. Since the rate of GTP hydrolysis is approximately 100 times slower than the rate of production of cAMP by the catalytic unit, one hormone± receptor complex is able to generate many cAMP molecules per minute. Another amplification step is between the receptor and the G protein, since the hormone ± receptor complex interacts transiently with the G protein and dissociates from it, once the G protein has been loaded with GTP. The receptor then interacts with other G protein molecules. The amplification factor of this step has been estimated to be about 10 in the -adrenergic-dependent adenylyl cyclase system. For the light-dependent activation of cyclic guanosine monophosphate (cGMP) dependent phosphodiesterase by rhodopsin, it was found that each activated molecule of rhodopsin activates approximately 300 phosphodiesterase molecules. This mechanism of activation is known as TMcollision coupling∫. Many other G-protein-coupled receptor systems have since been discovered, but the main features of the signaling pathway remain essentially similar to those initially described for hormone-dependent adenylyl cyclases. The prevailing dogma for the action of G-protein-coupled receptors is still based on the detailed biochemical studies performed on hormone-dependent adenylyl cyclase. The Dogma

Patterns of meiotic double-strand breakage on native and artificial yeast chromosomes

The preferred positions for meiotic double-strand breakage were mapped onSaccharomyces cerevisiae chromosomes I and VI, and on a number of yeast artificial chromosomes carrying human DNA inserts. Each chromosome had strong and weak double-strand break (DSB) sites. On average one DSB-prone region was detected by pulsed-field gel electrophoresis per 25 kb of DNA, but each chromosome had a unique distribution of DSB sites. There were no preferred meiotic DSB sites near the telomeres. DSB-prone regions were associated with all of the known “hot spots” for meiotic recombination on chromosomes I, III and VI.

Microwave-Assisted Solid-Phase Aza-peptide Synthesis: Aza Scan of a PKB/Akt Inhibitor Using Aza-arginine and Aza-proline Precursors

Aza-peptides are peptidomimetics in which one or more of the α-carbons, bearing the side-chain residues, has been replaced by a nitrogen. These peptidomimetics have been shown to be promising for the generation of drug leads and for structure-activity relationship studies. Aza-scan is the systematic replacement of amino acid residues in a given peptide with their aza counterparts. We report here an aza-scan of a potent, peptide-based PKB/Akt inhibitor, PTR6154. Procedures for microwave-assisted, Fmoc/t-Bu chemistry, solid-phase aza-peptide synthesis were developed which significantly reduce standard reaction time and are suitable for automation. Novel substituted hydrazines have been prepared for the straightforward incorporation of aza-arginine and aza-proline residues. This work will enable aza-scan to become a more common and standard method for structure-activity relationship studies of peptides.

DNA motif associated with meiotic double-strand break regions in Saccharomyces cerevisiae

Meiotic recombination in yeast is initiated by DNA double‐strand breaks (DSBs) that occur at preferred sites, distributed along the chromosomes. These DSB sites undergo changes in chromatin structure early in meiosis, but their common features at the level of DNA sequence have not been defined until now. Alignment of 1 kb sequences flanking six well‐mapped DSBs has allowed us to define a flexible sequence motif, the CoHR profile, which predicts the great majority of meiotic DSB locations. The 50 bp profile contains a poly(A) tract in its centre and may have several gaps of unrelated sequences over a total length of up to 250 bp. The major exceptions to the correlation between CoHRs and preferred DSB sites are at telomeric regions, where DSBs do not occur. The CoHR sequence may provide the basis for understanding meiosis‐induced chromatin changes that enable DSBs to occur at defined chromosomal sites.

Novel method for the synthesis of urea backbone cyclic peptides using new Alloc‐protected glycine building units

Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide, disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine functionalized side chains. Here we present urea backbone cyclization as an additional method for the preparation of backbone cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc‐Nα [ω‐amino(Alloc)‐alkyl] glycine building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for protein kinase B inhibition as anticancer leads. Copyright

Shift from apoptotic to necrotic cell death during human papillomavirus-induced transformation of keratinocytes.

Oncogenic transformation is a complex, multistep process, which goes through several stages before complete malignant transformation occurs. To identify early processes in carcinogenesis, we used an in vitro model, based on the initiating event in cervical cancer, papillomavirus transformation of keratinocytes. We compared gene expression in primary keratinocytes (K) and papillomavirus-transformed keratinocytes from early (E) and late (L) passages and from benzo[a]pyrene-treated L cells (BP). The transformed cells exhibit similar transcriptional changes to clinical cervical carcinoma. The number of transcripts expressed progressively decreased during the evolution from K to BP cells. Bioinformatic analysis, validated by detailed biochemical analysis, revealed substantial contraction of both pro- and antiapoptotic networks during transformation. Nonetheless, L and BP cells were not resistant to apoptotic stimuli. At doses of cisplatin that led to 30-60% apoptosis of K and E cells, transformed L and BP cells underwent 80% necrotic cell death, which became the default response to genotoxic stress. Moreover, appreciable necrotic fractions were observed in the cervical carcinoma cell line, HeLa, in response to comparable doses of cisplatin. The shrinkage of biochemical networks, including the apoptotic network, may allow a cancer cell to economize on energy usage to facilitate enhanced proliferation but leaves it vulnerable to stress. This study supports the hypothesis that the process of cancer transformation may be accompanied by a shift from apoptosis to necrosis.

Optimization of Energy-Consuming Pathways towards Rapid Growth in HPV-Transformed Cells

Cancer is a complex, multi-step process characterized by misregulated signal transduction and altered metabolism. Cancer cells divide faster than normal cells and their growth rates have been reported to correlate with increased metabolic flux during cell transformation. Here we report on progressive changes in essential elements of the biochemical network, in an in vitro model of transformation, consisting of primary human keratinocytes, human keratinocytes immortalized by human papillomavirus 16 (HPV16) and passaged repeatedly in vitro, and the extensively-passaged cells subsequently treated with the carcinogen benzo[a]pyrene. We monitored changes in cell growth, cell size and energy metabolism. The more transformed cells were smaller and divided faster, but the cellular energy flux was unchanged. During cell transformation the protein synthesis network contracted, as shown by the reduction in key cap-dependent translation factors. Moreover, there was a progressive shift towards internal ribosome entry site (IRES)-dependent translation. The switch from cap to IRES-dependent translation correlated with progressive activation of c-Src, an activator of AMP-activated protein kinase (AMPK), which controls energy-consuming processes, including protein translation. As cellular protein synthesis is a major energy-consuming process, we propose that the reduction in cell size and protein amount provide energy required for cell survival and proliferation. The cap to IRES-dependent switch seems to be part of a gradual optimization of energy-consuming mechanisms that redirects cellular processes to enhance cell growth, in the course of transformation.