Heterozygous ATM mutations do not contribute to early onset of breast cancer

Ataxia telangiectasia (AT) is a recessive syndrome, including cerebellar degeneration, immunologic defects and cancer predisposition, attributed to mutations in the recently isolated ATM (ataxia telangiectasia, mutated) gene. AT is diagnosed in 1/40,000 to 1/100,000 live births, with carriers calculated to comprise approximately 1% of the population. Studies of AT families have suggested that female relatives presumed to be carriers have a 5 to 8-fold increased risk for developing breast cancer, raising the possibility that germline ATM mutations may account for approximately 5% of all breast cancer cases. The increased risk for breast cancer reported for AT family members has been most evident among younger women, leading to an age-specific relative risk model predicting that 8% of breast cancer in women under age 40 arises in AT carriers, compared with 2% of cases between 40-59 years. To test this hypothesis, we undertook a germ-line mutational analysis of the ATM gene in a population of women with early onset of breast cancer, using a protein truncation (PTT) assay to detect chain-terminating mutations, which account for 90% of mutations identified in children with AT. We detected a heterozygous ATM mutation in 2/202 (1%) controls, consistent with the frequency of AT carriers predicted from epidemiologic studies. ATM mutations were present in only 2/401 (0.5%) women with early onset of breast cancer (P = 0.6). We conclude that heterozygous ATM mutations do not confer genetic predisposition to early onset of breast cancer.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma:analysis of a clinic-based population

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

BubR1 is required for the mitotic block induced by combretastatin-A4 and a novel cis-restricted ß-lactam analogue in human cancer cells

BubR1 is a well-defined guardian of the mitotic spindle, initiating mitotic arrest in response to the lack of tension and/or chromosome alignment across the mitotic plate. However, the role of BubR1 in combretastatin-induced cell death remains unknown. In this study, we describe the effects of combretastatin A-4 (CA-4) and a synthetic cis-restricted 3,4-diaryl-2-azetidinone (ß-lactam) analogue (CA-432) on the modulation and phosphorylation of BubR1 in human cervical cancer-derived cells. We demonstrate that CA-4 and CA-432 depolymerise the microtubular network of human cervical carcinoma-derived cells. Both compounds induced the disassembly of the microtubules and the loss of microtubule tension led to the early phosphorylation of BubR1 and the late cleavage of BubR1. The phosphorylation of BubR1 correlated with the onset of G2M cell cycle arrest whilst the cleavage of BubR1 coincided with apoptosis induced by the combretastatins. The combretastatin-induced apoptosis and the BubR1 cleavage were caspase-dependent. In vitro enzyme digests demonstrated that combretastatin-activated BubR1 is a substrate for caspase-3. Gene silencing of BubR1 with small interfering RNA severely compromised combretastatin-induced G2M cell cycle arrest with a corresponding increase in the formation of polyploid cells in both cervical and breast cancer-derived cells. In summary, BubR1 is required to maintain the G2M arrest and limit the formation of polyploid cells in response to continued combretastatin exposure. Moreover, substitution of the ethylene bridge with 3,4-diaryl-2-azetidinone did not alter the tubulin depolymerising properties or the subsequent mitotic spindle checkpoint response to CA-4 in human cancer cells.

BubR1 is required for a sustained mitotic spindle checkpoint arrest in human cancer cells treated with tubulin-targeting pyrrolo-1,5-benzoxazepines

Intrinsic or acquired resistance to chemotherapy is a major clinical problem that has evoked the need to develop innovative approaches to predict and ultimately reverse drug resistance. A prolonged G(2)M arrest has been associated with apoptotic resistance to various microtubule-targeting agents (MTAs). In this study, we describe the functional significance of the mitotic spindle checkpoint proteins, BubR1 and Bub3, in maintaining a mitotic arrest after microtubule disruption by nocodazole and a novel series of MTAs, the pyrrolo-1,5-benzoxazepines (PBOXs), in human cancer cells. Cells expressing high levels of BubR1 and Bub3 (K562, MDA-MB-231, and HeLa) display a prolonged G(2)M arrest after exposure to MTAs. On the other hand, cells with low endogenous levels of mitotic spindle checkpoint proteins (SK-BR-3 and HL-60) transiently arrest in mitosis and undergo increased apoptosis. The phosphorylation of BubR1 correlated with PBOX-induced G(2)M arrest in four cell lines tested, indicating an active mitotic spindle checkpoint. Gene silencing of BubR1 by small interfering RNA interference reduced PBOX-induced G(2)M arrest without enhancing apoptotic efficacy. Further analysis demonstrated that PBOX-treated BubR1-depleted cells were both mononucleated and multinucleated with a polyploid DNA content, suggesting a requirement for BubR1 in cytokinesis. Taken together, these results suggest that BubR1 contributes to the mitotic checkpoint induced by the PBOXs.

Selective induction of apoptosis by the pyrrolo-1,5-benzoxazepine 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) in Leukemia cells occurs via the c-Jun NH2-terminal kinase-dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL

Overexpression of the Bcl-2 proto-oncogene in tumor cells confers resistance against chemotherapeutic drugs. In this study, we describe how the novel pyrrolo-1,5-benzoxazepine compound 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) selectively induces apoptosis in Bcl-2-overexpressing cancer cells, whereas it shows no cytotoxic effect on normal peripheral blood mononuclear cells. PBOX-6 overcomes Bcl-2-mediated resistance to apoptosis in chronic myelogenous leukemia (CML) K562 cells by the time- and dose-dependent phosphorylation and inactivation of antiapoptotic Bcl-2 family members Bcl-2 and Bcl-XL. PBOX-6 also induces Bcl-2 phosphorylation and apoptosis in wild-type T leukemia CEM cells and cells overexpressing Bcl-2. This is in contrast to chemotherapeutic agents such as etoposide, actinomycin D, and ultraviolet irradiation, whereby overexpression of Bcl-2 confers resistance against apoptosis. In addition, PBOX-6 induces Bcl-2 phosphorylation and apoptosis in wild-type Jurkat acute lymphoblastic leukemia cells and cells overexpressing Bcl-2. However, Jurkat cells containing a Bcl-2 triple mutant, whereby the principal Bcl-2 phosphorylation sites are mutated to alanine, demonstrate resistance against Bcl-2 phosphorylation and apoptosis. PBOX-6 also induces the early and transient activation of c-Jun NH2-terminal kinase (JNK) in CEM cells. Inhibition of JNK activity prevents Bcl-2 phosphorylation and apoptosis, implicating JNK in the upstream signaling pathway leading to Bcl-2 phosphorylation. Collectively, these findings identify Bcl-2 phosphorylation and inactivation as a critical step in the apoptotic pathway induced by PBOX-6 and highlight its potential as an effective antileukemic agent.

Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays:towards new standards for gene expression measurements

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic myelogenous leukemia (CML) cells by bypassing the apoptotic suppressor bcr-abl

Expression of the transforming oncogene bcr-abl in chronic myelogenous leukemia (CML) cells is reported to confer resistance against apoptosis induced by many chemotherapeutic agents such as etoposide, ara-C, and staurosporine. In the present study some members of a series of novel pyrrolo-1,5-benzoxazepines potently induce apoptosis, as shown by cell shrinkage, chromatin condensation, DNA fragmentation, and poly(ADP-ribose) polymerase (PARP) cleavage, in three CML cell lines, K562, KYO.1, and LAMA 84. Induction of apoptosis by a representative member of this series, PBOX-6, was not accompanied by either the down-regulation of Bcr-Abl or by the attenuation of its protein tyrosine kinase activity up to 24 h after treatment, when approximately 50% of the cells had undergone apoptosis. These results suggest that down-regulation of Bcr-Abl is not part of the upstream apoptotic death program activated by PBOX-6. By characterizing the mechanism in which this novel agent executes apoptosis, this study has revealed that PBOX-6 caused activation of caspase 3-like proteases in only two of the three CML cell lines. In addition, inhibition of caspase 3-like protease activity using the inhibitor z-DEVD-fmk blocked caspase 3-like protease activity but did not prevent the induction of apoptosis, suggesting that caspase 3-like proteases are not essential in the mechanism by which PBOX-6 induces apoptosis in CML cells. In conclusion, this study demonstrates that PBOX-6 can bypass Bcr-Abl-mediated suppression of apoptosis, suggesting an important potential use of these compounds in the treatment of CML.

Autosomal dominant retinitis pigmentosa:a new multi-allelic marker (D3S621) genetically linked to the disease locus (RP4)

We report the characterization of a new eight-allele microsatellite (D3S621) isolated from a human chromosome 3 library. Two-point and multi-locus genetic linkage analysis have shown D3S621 to co-segregate with the previously mapped RP4 (theta m = 0.12, Zm = 4.34) and with other genetic markers on the long arm of the chromosome, including D3S14 (R208) (theta m = 0.00, Zm = 15.10), D3S47 (C17) (theta m = 0.11, Zm = 4.95), Rho (theta m = 0.07, Zm = 1.37), D3S21 (L182) (theta m = 0.07, Zm = 2.40) and D3S19 (U1) (theta m = 0.13, Zm = 2.78). This highly informative marker, with a polymorphic information content of 0.78, should be of considerable value in the extension of linkage data for autosomal dominant retinitis pigmentosa with respect to locii on the long arm of chromosome 3.

Quantification of Type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

The Gram-negative bacterial type VI Secretion System (T6SS) delivers toxins to kill orinhibit the growth of susceptible bacteria, while others target eukaryotic cells. Deletionof atsR, a negative regulator of virulence factors in B. cenocepacia K56-2, increasesT6SS activity. Macrophages infected with a K56-2 ΔatsR mutant display dramaticalterations in their actin cytoskeleton architecture that rely on the T6SS, which isresponsible for the inactivation of multiple Rho-family GTPases by an unknownmechanism. We employed a strategy to standardize the bacterial infection ofmacrophages and densitometrically quantify the T6SS-associated cellular phenotype,which allowed us to characterize the phenotype of systematic deletions of each genewithin the T6SS cluster and ten vgrG encoding genes in K56-2 ΔatsR. None of thegenes from the T6SS core cluster and the individual vgrGs were directly responsiblefor the cytoskeletal changes in infected cells. However, a mutant strain with all vgrGgenes deleted was unable to cause macrophage alterations. Despite not being able toidentify a specific effector protein responsible for the cytoskeletal defects inmacrophages, our strategy resulted in the identification of the critical core componentsand accessory proteins of the T6SS assembly machinery and provides a screeningmethod to detect T6SS effectors targeting the actin cytoskeleton in macrophages byrandom mutagenesis.

Endosomal signaling and oncogenesis

The endosomal system provides a route whereby nutrients, viruses, and receptors are internalized. During the course of endocytosis, activated receptors can accumulate within endosomal structures and certain signal-transducing molecules can be recruited to endosomal membranes. In the context of signaling and cancer, they provide platforms within the cell from which signals can be potentiated or attenuated. Regulation of the duration of receptor signaling is a pivotal means of refining growth responses in cells. In cancers, this is often considered in terms of mutations that affect receptor tyrosine kinases and maintain them in hyperactivated states of dimerization and/or phosphorylation. However, disruption to the regulatory control exerted by the assembly of protein complexes within the endosomal network can also contribute to disease among which oncogenesis is characterized in part by dysregulated growth, enhanced cell survival, and changes in the expression of markers of differentiation. In this chapter, we will discuss the role of proteins that regulate in endocytosis as tumor suppressors or oncogenes and how changing the fate of internalized receptors and concomitant endosomal signaling can contribute to cancer.

Nuclear translocation and functions of growth factor receptors

Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?

AURKA overexpression accompanies dysregulation of DNA-damage response genes in invasive urothelial cell carcinoma

Invasive urothelial cell carcinoma (UCC) is characterized by increased chromosomal instability and follows an aggressive clinical course in contrast to non-invasive disease. To identify molecular processes that confer and maintain an aggressive malignant phenotype, we used a high-throughput genome-wide approach to interrogate a cohort of high and low clinical risk UCC tumors. Differential expression analyses highlighted cohesive dysregulation of critical genes involved in the G(2)/M checkpoint in aggressive UCC. Hierarchical clustering based on DNA Damage Response (DDR) genes separated tumors according to a pre-defined clinical risk phenotype. Using array-comparative genomic hybridization, we confirmed that the DDR was disrupted in tumors displaying high genomic instability. We identified DNA copy number gains at 20q13.2-q13.3 (AURKA locus) and determined that overexpression of AURKA accompanied dysregulation of DDR genes in high risk tumors. We postulated that DDR-deficient UCC tumors are advantaged by a selective pressure for AURKA associated override of M phase barriers and confirmed this in an independent tissue microarray series. This mechanism that enables cancer cells to maintain an aggressive phenotype forms a rationale for targeting AURKA as a therapeutic strategy in advanced stage UCC.

The developing role of receptors and adaptors

The response of a cell to the myriad of signals that it receives is varied, and it is dependent on many different factors. The most-studied responses involve growth-factor signalling and these signalling cascades have become key targets for cancer therapy. Recent reports have indicated that growth-factor receptors and associated adaptors can accumulate in the nucleus. Are there novel functions for these proteins that might affect our understanding of their role in cancer and have implications for drug resistance?

Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.