Implications of CLSPN Variants in Cellular Function and Susceptibility to Cancer.

Claspin is a multifunctional protein that participates in physiological processes essential for cell homeostasis that are often defective in cancer, namely due to genetic changes. It is conceivable that Claspin gene (CLSPN) alterations may contribute to cancer development. Therefore, CLSPN germline alterations were characterized in sporadic and familial breast cancer and glioma samples, as well as in six cancer cell lines. Their association to cancer susceptibility and functional impact were investigated. Eight variants were identified (c.-68C>T, c.17G>A, c.1574A>G, c.2230T>C, c.2028+16G>A, c.3595-3597del, and c.3839C>T). CLSPN c.1574A>G (p.Asn525Ser) was significantly associated with breast cancer and was shown to cause partial exon skipping and decreased Claspin expression and Chk1 activation in a minigene splicing assay and in signalling experiments, respectively. CLSPN c.2028+16G>A was significantly associated with familial breast cancer and glioma, whereas c.2230T>C (p.Ser744Pro), was exclusively detected in breast cancer and glioma patients, but not in healthy controls. The remaining variants lacked a significant association with cancer. Nevertheless, the c.-68C>T promoter variant increased transcriptional activity in a luciferase assay. In conclusion, some of the CLSPN variants identified in the present study appear to modulate Claspin's function by altering CLSPN transcription and RNA processing, as well as Chk1 activation.

Claspin: From replication stress and DNA damage responses to cancer therapy.

Cancer is still one of the major causes of death worldwide. Radiation therapy and chemotherapy remain the main treatment modalities in cancer. These therapies exert their effect mainly through interference with DNA replication and induction of DNA damage. It is believed that one way of improving the efficacy of cancer treatment will be to inhibit the replication stress and DNA damage responses and promote mitotic catastrophe of cancer cells. So far, the majority of the efforts have focused central players of checkpoint responses, such as ATR and CHK1, and DNA damage repair, such as PARPs. Being a key player in the replication stress response, checkpoint activation, and the DNA damage response, Claspin constitutes an attractive therapeutic target in cancer, namely for radio- and chemo-sensitization. In this review, we will go through Claspin functions in the replication stress and DNA damage responses and will discuss how Claspin can be targeted in cancer treatment, as well as the effects of Claspin inhibition.

Claspin functions in cell homeostasis-A link to cancer?

Cancer remains one of the leading causes of mortality worldwide. Most cancers present high degrees of genomic instability. DNA damage and replication checkpoints function as barriers to halt cell cycle progression until damage is resolved, preventing the perpetuation of errors. Activation of these checkpoints is critically dependent on Claspin, an adaptor protein that mediates the phosphorylation of the effector kinase Chk1 by ATR. However, Claspin also performs other roles related to the protection and maintenance of cell and genome integrity. For instance, following DNA damage and checkpoint activation, Claspin bridges checkpoint responses to DNA repair or to apoptosis. During DNA replication, Claspin acts a sensor and couples DNA unwinding to strand polymerization, and may also indirectly regulate replication initiation at firing origins. As Claspin participates in several processes that are vital to maintenance of cell homeostasis, its function is tightly regulated at multiple levels. Nevertheless, little is known about its role in cancer. Accumulating evidence suggests that Claspin inactivation could be an essential event during carcinogenesis, indicating that Claspin may function as a tumour suppressor. In this review, we will examine the functions of Claspin and how its deregulation may contribute to cancer initiation and progression. To conclude, we will discuss means by which Claspin can be targeted for cancer therapy.

Adult Attachment and Dyadic Adjustment: The Mediating Role of Shame.

Although it is widely recognized that adult attachment is associated with romantic relationship quality, the mechanisms involved remain poorly understood. This study aimed to investigate the mediating role of external and internal shame on the association between attachment and dyadic adjustment. A battery of self-report measures was completed by 228 Portuguese participants and a serial multiple mediation model was tested. Data showed that, in the population under study, attachment dimensions were associated with worse dyadic adjustment through high external and internal shame. Internal shame alone also mediated the association between attachment avoidance and dyadic adjustment. This study identifies a new putative mechanism linking adult attachment and intimate relationship functioning that may be targeted in couples therapy to promote a better dyadic adjustment and relationship functioning.

Adult attachment insecurity and dyadic adjustment: The mediating role of self-criticism.

OBJECTIVES: Although it is well established that adult attachment is associated with relationship quality, the mechanisms involved in this association are still poorly understood. Individual variables that are shaped in early attachment experiences, such as self-criticism, may be particularly important. The present study aimed to investigate the mediating role of self-criticism and self-reassurance on the association between attachment-related anxiety and avoidance and dyadic adjustment. DESIGN AND METHODS: About 230 individuals from a community sample completed an online battery of self-report measures of adult attachment, dyadic adjustment, and forms of self-criticism and self-reassuring. A parallel mediation model was tested. RESULTS: Data showed that attachment anxiety and avoidance were associated with poorer dyadic adjustment through high levels of self-criticism in the form of an inadequate self. CONCLUSION: Our findings highlight the importance of targeting feelings of self-inadequacy in couple therapy to promote better dyadic adjustment and relationship functioning. The innovative contribution of this work is the identification of a new mechanism underlying the association between adult attachment and dyadic functioning. PRACTITIONER POINTS: Self-criticism in the form of an inadequate self mediates the association between attachment and dyadic adjustment. Although correlated with attachment dimensions and dyadic adjustment, the hated self and the reassured self do not act as mediators of the relationship between attachment and dyadic adjustment. It seems important to evaluate and address feelings of inadequacy in the context of couple therapy.

In vitro transforming potential, intracellular signaling properties, and sensitivity to a kinase inhibitor (sorafenib) of RET proto-oncogene variants Glu511Lys, Ser649Leu, and Arg886Trp.

Multiple endocrine neoplasia type 2 and a subset of apparently sporadic medullary thyroid carcinoma (AS-MTC) are caused by germ line activating point mutations of the rearranged during transfection (RET) proto-oncogene. RET encodes a receptor with tyrosine kinase activity that targets several intracellular signaling cascades, such as RAS-RAF-ERK1/2, PIK3-AKT, and STAT transcription factors. The objective of this study was to assess the function of three germ line RET variants Arg886Trp, Ser649Leu, and Glu511Lys of undetermined pathogenic significance, which were found in three kindreds of isolated AS-MTC. For this purpose, we employed vectors expressing each of the RET variants and measured the number of NIH3T3 transformation foci and soft agar colonies, the degree of activation of known RET intracellular signaling targets (ERK1/2, STAT1, STAT3, and TCF4), and the extent of ERK1/2 inhibition on sorafenib treatment. We found that RET variants Arg886Trp and Glu511Lys have shown increased in vitro transforming potential in a glial-derived neurotrophic factor-dependent manner. In contrast, the Ser649Leu variant did not significantly increased the number of foci and agar colonies relative to wild-type RET (RET-WT). The variants Glu511Lys and Arg886Trp showed 10- and 12.5-fold ERK1/2 activation respectively, that was significantly higher than that observed for RET-WT (fivefold). Increased levels of STAT1 and TCF4 activation were only observed for RET Arg886Trp (2.5- and 3-fold versus 1.2- and 2-fold in RET-WT respectively). The three RET variants analyzed here were sensitive to treatment with sorafenib. In conclusion, our results allow to classify previously uncharacterized RET genotypes, which may be of use to define follow-up and therapeutic regimens.

Knocking out of CD38 accelerates development of a lupus-like disease in lpr mice.

OBJECTIVES: CD38 participates in lymphocyte ontogeny and function and may be involved in autoimmunity. Absence of CD38 accelerates development of non-obese diabetic (NOD) mice diabetes and anti-CD38 antibodies are good markers of human disease. Little is known regarding systemic autoimmunity. Active SLE patients have higher numbers of CD38(+) T and B cells. CD38 is a candidate gene for the murine Lmb2 lupus locus. We aimed to investigate whether CD38 was involved in lupus development. METHODS: We developed Cd38(-/-)-Fas(lpr)/Fas(lpr) mice and monitored them for development of a lupus-like disease through measurement of protein excretion in urine, histological assessment of the kidneys, quantification of circulating immunoglobulins and autoantibodies. We have also immunophenotyped 2- and 6-month old Cd38(-/-)-Fas(lpr)/Fas(lpr) mice. RESULTS: We found that absence of CD38 accelerated disease development: female Cd38(-/-)-Fas(lpr)/Fas(lpr) mice presented severe proteinuria, GN, deposition of ICs in the renal medulla and increased amounts of circulating immunoglobulin G (IgG), although anti-dsDNA autoantibodies and RF were not significantly increased at disease onset. We have found that Cd38(-/-)-Fas(lpr)/Fas(lpr) male mice, similarly to other murine models of lupus, were able to control disease. Absence of CD38 in lpr mice altered differentiation of T cells and dendritic cells (DC). CONCLUSION: Although the role of CD38 in tolerance is still to be elucidated, we provide evidence that it may play an active role in the control of a murine lupus-like disease.

Characterization of common and rare human papillomaviruses in Portuguese women by the polymerase chain reaction, restriction fragment length polymorphism and sequencing.

The present study aimed to provide additional information on the prevalence of mucosal human papillomavirus (HPV) types in Portuguese women by using polymerase chain reaction, restriction fragment length polymorphism and sequencing. HPV was detected in 15.5% (15/97) of the control samples, 23.5% (12/51) of atypical squamous cells of undetermined significance, 52.8% (28/53) of low-grade lesions, 82.4% (28/34) of high-grade lesions, and 100% (44/44) of carcinomas. Overall, 28 HPV types were detected: 11 high-risk (HPV 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, and 59), 3 probable high-risk (HPV 53, 66, and 73), 6 low-risk (HPV 6, 11, 44, 61, 70, and 81), and 8 unknown-risk types (HPV 34, 62, 67, 71, 83, 84, 102, and 108). The most prevalent type was HPV 16, detected in 33.8% of women infected with HPV, followed by HPV 58 (9.2%), HPV 33 (7.0%), HPV 18 (6.3%), HPV 53 (5.6%), HPV 31 and 56 (4.9% each), HPV 6 (3.5%), and HPV 66 and 81 (2.8% each). Of 44 cervical carcinoma samples, 71% were associated with HPV 16 (60%) and HPV 18 (11.1%), followed by the high-risk types 33 (11.1%), 35 (4.4%), 45 (4.4%), and 56 (2.2%), the probable high-risk type 53 (4.4%) and the unknown-risk type 67 (2.2%). This study provides information on the most common HPV types in Portuguese women and suggests that the current prophylactic HPV 16/18 vaccine may be useful for the prevention of cervical cancer in this population.

Complete genotyping of mucosal human papillomavirus using a restriction fragment length polymorphism analysis and an original typing algorithm.

BACKGROUND: Due to the differences in the oncogenic activity of human papillomaviruses (HPV), it is clinically important to accurately identify HPV types in a simple and time effective manner. OBJECTIVES: We aimed at developing a straightforward and cost-effective assay to individually identify all mucosal HPVs, based on the amplification of L1 gene using MY09/11 primers, and subsequent restriction fragment length polymorphism (RFLP) analysis. STUDY DESIGN: We made use of bioinformatic tools to analyze all published DNA sequences of 49 mucosal HPV types for PstI, HaeIII, DdeI and RsaI restriction sites. Based on the RFLP patterns, we have designed an original genotyping algorithm. RESULTS: Each HPV type presented a distinct RFLP pattern, which was visually distinguishable on polyacrylamide gels. A set of 27 pre-selected patient samples of known HPV types was confirmed positive for the same HPV type using this RFLP assay. Furthermore, in a random and blind HPV typing experiment performed in 30 untyped clinical samples, RFLP data consistently matched DNA sequencing results. CONCLUSIONS: Our polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, using 4 restriction enzymes (PstI, HaeIII, DdeI, RsaI) and an original genotyping algorithm, allows discrimination of all individual mucosal HPV types in single infections, and even detection of multiple infections. This assay gives complementary information to commercially available methods, and may also be financially advantageous, particularly when financial resources are scarce.

Loss of heterozygosity at 19p13.2 and 2q21 in tumours from familial clusters of non-medullary thyroid carcinoma.

Linkage studies have identified susceptibility loci for familial nonmedullary thyroid cancer (FNMTC), with and without cell oxyphilia, at chromosomal regions 19p13.2 and 2q21. There are few genetic analyses of FNMTC tumours reported at the present time and the eventual gene involved was not identified yet. The aim of this study was to assess the occurrence of loss of heterozygosity (LOH) at these loci in the tumours from familial clusters of NMTC. We have analysed LOH in 14 tumours from 9 two-case familial clusters of NMTC. Using paired blood (normal) and tumour DNA samples, we have genotyped ten microsatellite and one SNP markers throughout 19p13.2 and fourteen microsatellite markers at 2q21. Overall, eight (57%) and two (14%) out of the fourteen tumours analysed exhibited LOH at 19p13.2 and 2q21, respectively. In two families (22%), LOH for the same markers was demonstrable in the tumours of the two members of the same family. In one family (11%) LOH was demonstrable at both loci analysed. In four two-case familial clusters (44%), LOH at the 19p13.2 locus was found in only one of the tumour cases analysed. Detailed haplotype analysis showed that, in two families (22%), the pattern of LOH in tumours was consistent with selective retention of the haplotype shared by affected members. In the remaining cases, it was consistent with random allelic losses. In conclusion, we report the finding of LOH at the 19p13.2 and 2q21 loci in tumours from familial clusters of NMTC, providing evidence that inactivation of putative genes in these regions, acting as tumour-suppressors, may be involved in the development of tumours in the context of FNMTC.

CD38 plays a role in effective containment of mycobacteria within granulomata and polarization of Th1 immune responses against Mycobacterium avium.

CD38 is a multifunctional ectoenzyme that behaves either as an enzyme, a cell adhesion molecule or as a cell surface receptor involved in cell signalling. It is expressed in cells of several lineages, including B and T lymphocytes, and macrophages. CD38 was shown to be important for the development of T-cell dependent humoral immune responses against extracellular pathogens. It also appears to be functionally important in macrophages, which are the host cells of Mycobacterium avium, an intracellular parasite that survives within these cells by avoiding a number of their microbicidal strategies. The present work aimed at investigating whether CD38 had any role on the immune response against mycobacterial infection. After intraperitoneal M. avium infection, the immune response of CD38KO mice was compared to that of their parental strain, C57Bl.6 mice. Absence of CD38 rendered mice more susceptible to mycobacterial infection. This susceptibility seems to be due to ineffective Th1 differentiation and polarization, which is essential for the control of M. avium infection. In addition, absence of CD38 seems to compromise the maintenance of the granulomatous barrier, leading to dissemination and unrestrained growth of mycobacteria.

Occurrence of the Cys611Tyr mutation and a novel Arg886Trp substitution in the RET proto-oncogene in multiple endocrine neoplasia type 2 families and sporadic medullary thyroid carcinoma cases originating from the central region of Portugal.

OBJECTIVE: Medullary thyroid carcinoma (MTC) occurs both sporadically and in the context of autosomal dominantly inherited multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC), which are caused by activating germline mutations in the RET proto-oncogene. The aim of this study was to characterize the RET mutational spectrum in MEN2 families and apparently sporadic MTC (AS-MTC) cases originating from the central region of Portugal. SUBJECTS AND METHODS: We studied a total of 82 individuals (64 affected and 18 family members), comprising five MEN2 families (four MEN2A and one MEN2B), as well as 53 AS-MTC cases. RET germline mutations were screened using PCR-DNA sequencing, SSCP and RFLP. The haplotypes associated with recurrent mutations were determined by fragment analysis of microsatellite markers, and by RFLP, in the case of intragenic polymorphisms. RESULTS: Frequency of the Cys611Tyr (TGC-TAC) mutation was significantly increased in this region of Portugal, due to the fact that three apparently unrelated MEN2A/FMTC families, out of the five in which mutations were identified, harboured this specific mutation. Haplotype analysis revealed that a common haplotype was shared between two of these three families. We have also characterized a novel RET mutation, Arg886Trp, located in the tyrosine kinase domain, which was found in an AS-MTC case. CONCLUSIONS: There are regional specificities in the relative frequency of RET mutations, which are consistent with a cluster-like distribution of specific disease-causing mutations, as a result of the inheritance of a shared haplotype. These data, along with the finding of a novel RET mutation (Arg886Trp), have important implications towards facilitating and improving the molecular diagnosis of hereditary MTC on a regional basis.