Islet cells in human type 1 diabetes: from recent advances to novel therapies - a symposium-based roadmap for future research.

There is a growing understanding that the early phases of type 1 diabetes (T1D) are characterised by a deleterious dialogue between the pancreatic beta cells and the immune system. This, combined with the urgent need to better translate this growing knowledge into novel therapies, provided the background for the JDRF-DiabetesUK-INNODIA-nPOD symposium entitled 'Islet cells in human T1D: from recent advances to novel therapies', which took place in Stockholm, Sweden, in September 2022. We provide in this article an overview of the main themes addressed in the symposium, pointing to both promising conclusions and key unmet needs that remain to be addressed in order to achieve better approaches to prevent or reverse T1D.

Identification of ubiquitin ligases required for skeletal muscle atrophy.

Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.

The de-ubiquitinating enzyme Unp interacts with the retinoblastoma protein.

The ubiquitin pathway is involved in the proteolytic turnover of many short-lived cellular regulatory proteins. Since selective degradation of substrates of this system requires the covalent attachment of a polyubiquitin chain to the substrates, degradation could be counteracted by de-ubiquitinating enzymes (or isopeptidases) which selectively remove the polyubiquitin chain. Unp is a human isopeptidase with still poorly understood biological functions. Here, we show that cellular Unp specifically interacts with the retinoblastoma gene product (pRb).

Induction of beta-transducin repeat-containing protein by JNK signaling and its role in the activation of NF-kappaB.

Activation of Jun N-kinase (JNK) and NF-kappaB transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-kappaB inhibitor (IkappaB) by respective stress-inducible kinases (IKK) is a key event in NF-kappaB activation. beta-TrCP F-box protein mediates ubiquitination of phosphorylated IkappaB via recruitment of SCF(beta-TrCP)-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IkappaB results in activation of the NF-kappaB pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of beta-TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKKbeta) also leads to accumulation of beta-TrCP. Transcription of the beta-TrCP gene is not required for JNK-mediated induction of beta-TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-kappaB was observed. The mechanisms of beta-TrCP induction via stress and its role in NF-kappaB activation are discussed.

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d).

Male mice lacking both the Ink4c and Ink4d genes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18(Ink4c) and p19(Ink4d) are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking both Ink4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lacking Ink4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast, Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed in Ink4c/Ink4d double-null males. Our data indicate that p18(Ink4c) and p19(Ink4d) are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.

Role of the F-box protein Skp2 in lymphomagenesis.

The F-box protein Skp2 (S-phase kinase-associated protein 2) positively regulates the G(1)-S transition by controlling the stability of several G(1) regulators, such as the cell cycle inhibitor p27. We show here that Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. To directly evaluate the potential of Skp2 to deregulate growth in vivo, we generated transgenic mice expressing Skp2 targeted to the T-lymphoid lineage as well as double transgenic mice coexpressing Skp2 and activated N-Ras. A strong cooperative effect between these two transgenes induced T cell lymphomas with shorter latency and higher penetrance, leading to significantly decreased survival when compared with control and single transgenic animals. Furthermore, lymphomas of Nras single transgenic animals often expressed higher levels of endogenous Skp2 than tumors of double transgenic mice. This study provides evidence of a role for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas.

Limited overlapping roles of P15(INK4b) and P18(INK4c) cell cycle inhibitors in proliferation and tumorigenesis.

Entry of quiescent cells into the cell cycle is driven by the cyclin D-dependent kinases Cdk4 and Cdk6. These kinases are negatively regulated by the INK4 cell cycle inhibitors. We report the generation of mice defective in P15(INK4b) and P18(INK4c). Ablation of these genes, either alone or in combination, does not abrogate cell contact inhibition or senescence of mouse embryo fibroblasts in culture. However, loss of P15(INK4b), but not of P18(INK4c), confers proliferative advantage to these cells and makes them more sensitive to transformation by H-ras oncogenes. In vivo, ablation of P15(INK4b) and P18(INK4c) genes results in lymphoproliferative disorders and tumor formation. Mice lacking P18(INK4c) have deregulated epithelial cell growth leading to the formation of cysts, mostly in the cortical region of the kidneys and the mammary epithelium. Loss of both P15(INK4b) and P18(INK4c) does not result in significantly distinct phenotypic manifestations except for the appearance of cysts in additional tissues. These results indicate that P15(INK4b) and P18(IKN4c) are tumor suppressor proteins that act in different cellular lineages and/or pathways with limited compensatory roles.

The human F box protein beta-Trcp associates with the Cul1/Skp1 complex and regulates the stability of beta-catenin.

Ubiquitin-conjugation targets numerous cellular regulators for proteasome-mediated degradation. Thus, the identification of ubiquitin ligases and their physiological substrates is crucially important, especially for those cases in which aberrant levels of regulatory proteins (e.g., beta-catenin, p27) result from a deregulated ubiquitination pathway. In yeast, the proteolysis of several G1 regulators is controlled by ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins. Specific F-box proteins (Fbps) recruit different substrates to the SCF. Although many Fbps have been identified in mammals, their specific substrates and the existence of multiple SCFs have not yet been reported. We have found that one human Fbp, beta-Trcp (beta-Transducin repeat containing protein), does indeed form a novel SCF with human Skp1 and Cul1. Consistent with recent reports indicating that Xenopus and Drosophila beta-Trcp homologs act as negative regulators of the Wnt/beta-catenin signaling pathway, we report here that human beta-Trcp interacts with beta-catenin in vivo. Furthermore, beta-catenin is specifically stabilized in vivo by the expression of a dominant negative beta-Trcp. These results indicate that the Cul1/Skp1/beta-Trcp complex forms a ubiquitin ligase that mediates the degradation of beta-catenin.

Cyclin-dependent kinase inhibitor p57KIP2 in soft tissue sarcomas and Wilms'tumors.

Mammalian cyclin-dependent kinase inhibitors fall into two families, the INK4 and the CIP/KIP. The CIP/KIP family comprises three structurally related members, including p21CiP1/WAF1, p27KIP1, and p57KIP2. These proteins are all capable of inhibiting the progression of the cell cycle by binding and inhibiting G(1) cyclin/cyclin-dependent kinase complexes. In humans, p57KIP2 is expressed specifically in skeletal muscle, heart, brain, kidney, and lung. Human KIP2 resides in 11p15.5, a chromosomal region that is a common site for loss of heterozygosity in certain sarcomas, Wilms' tumors, and tumors associated with the Beckwith-Wiedemann syndrome. Because of the function, selective expression, and chromosomal location of p57KIP2, we undertook the present study to search for potential mutations of KIP2 in a cohort of 126 tumors composed of 75 soft tissue sarcomas and 51 Wilms' tumors. The KIP2 gene was characterized by Southern blot, comparative multiplex PCR, PCR -single-strand conformational polymorphism, and DNA sequencing assays in these neoplasms. Deletions of the KIP2 gene or point mutations at the region encoding the cyclin-dependent kinase inhibitory domain were not found in the tumors analyzed. The absence of KIP2 mutations might indicate that these tumors arise due to defects at a closely linked but separate locus. Alternatively, similarly to the mouse homologue, inactivation of KIP2 could occur via genomic imprinting.

Altered patterns of retinoblastoma gene product expression in adult soft-tissue sarcomas.

Altered expression of the retinoblastoma (RB) tumour-suppressor gene product (pRB) has been detected in sporadic bone and soft-tissue sarcomas. Earlier studies, analysing small cohorts of sarcoma patients, have suggested that these alterations are more commonly associated with high-grade tumours, metastatic lesions and poorer survival. This study was designed to re-examine the prevalence and clinical significance of altered pRB expression in a large and selected group of soft-tissue sarcomas from 174 adult patients. Representative tissue sections from these sarcomas were analysed by immunohistochemistry using a well-characterised anti-pRB monoclonal antibody. Tumours were considered to have a positive pRB phenotype only when pure nuclear staining was demonstrated, and cases were segregated into one of three groups. Group 1 (n = 36) were patients whose tumours have minimal or undetectable pRB nuclear staining (< 20% of tumour cells) and were considered pRB negative. Patients with tumours staining in a heterogeneous pattern (20-79% of tumour cells) were classified as group 2 (n = 99). The staining of group 3 (n = 39) was strongly positive with a homogeneous pRB nuclear immunoreactivity (80-100% of tumour cells). pRB alterations were frequently observed in both low- and high-grade lesions. Altered pRB expression did not correlate with known predictors of survival and was not itself an independent predictor of outcome in the long-term follow-up. These findings support earlier observations that alterations of pRB expression are common events in soft-tissue sarcomas; nevertheless, long-term follow-up results indicate that altered patterns of pRB expression do not influence clinical outcome of patients affected with soft-tissue sarcomas. It is postulated that RB alterations are primary events in human sarcomas and may be involved in tumorigenesis or early phases of tumour progression in these neoplasias.

Genetic evidence in melanoma and bladder cancers that p16 and p53 function in separate pathways of tumor suppression.

The 9p21 region of human chromosome 9 is a hot spot for chromosomal aberrations in both cultured cell lines and primary tumors. This region contains a gene, P16 (also called MTS1, CDKN2 and p16INK4), that encodes a presumptive negative cell cycle regulator called p16. P16 is deleted or mutated at high frequency in a variety of tumor cell lines including melanoma and bladder carcinoma lines. As such, it is likely to be a tumor suppressor gene. Here we show that P16 is mutated in primary bladder carcinomas (3 of 33) and melanomas (5 of 34). These findings support studies that show P16 mutations are not solely a product of growth in tissue culture but rather are involved in formation of tumors in viva. Some bladder primary tumors and some bladder and melanoma tumor cell lines contain mutations in both P16 and P53 at frequencies that suggest that p53 and p16 function in different pathways, each of which is important in suppressing malignant transformation.

p27Kip1: chromosomal mapping to 12p12-12p13.1 and absence of mutations in human tumors.

The p27Kip1 gene codes for a cyclin-dependent kinase inhibitor implicated in G1 arrest by transforming growth factor beta, cell-cell contact, agents that elevate cyclic AMP, and the growth-inhibitory drug rapamycin. p27 binds to and inhibits complexes formed by cyclin E-cdk2, cyclin A-cdk2, and cyclin D-cdk4. The involvement of p27 in the negative regulation of cell proliferation suggests that it may also function as a tumor suppressor gene. Using a combination of somatic cell hybrid panels and fluorescence in situ hybridization p27Kip1 has been mapped to the short arm of chromosome 12 at the 12p12-12p13.1 boundary, reported to harbor deletions and rearrangements in leukemia and mesotheliomas. In order to assess potential p27Kip1 gene alterations, we have screened a total of 147 human primary solid tumors and found no detectable cancer-specific mutations. These results argue that the often observed loss of antimitogenic transforming growth factor beta responsiveness in human cancer cells is not due to structural defects in p27Kip1.

A new polymorphic site in intron 2 of TP53 characterizes LOH in human tumors by PCR-SSCP.

Many human cancers present deletions of the short arm of chromosome 17, which includes the TP53 locus. We detected a new polymorphism in intron 2 of the TP53 gene using PCR-SSCP and used this polymorphic site as a marker to detect loss of heterozygosity in 135 human tumors (73 soft tissue sarcomas, and 48 colorectal and 14 bladder carcinomas). Heterozygosity for this site was 41.5% in this study group and tumor-specific loss of alleles occurred in 43% of informative cases. Allelic losses were more frequently detected at this site than at that in which restriction fragment length polymorphism (RFLP) is located, as detected by the pHp53B probe. It is concluded that this novel approach has several advantages, including detection of a high incidence of informative cases and minimal tissue requirements.

Chromosome 17 abnormalities and TP53 mutations in adult soft tissue sarcomas.

This study was designed to determine the frequency of structural genetic abnormalities of chromosome 17 and the incidence of TP53 mutations as they relate to the biological behavior of adult soft tissue sarcomas. We analyzed a group of 73 soft tissue sarcomas of adults that were clinically and pathologically well characterized using molecular genetic techniques and expression studies. We then correlated genotype and phenotype with pathological parameters. Overall, allelic loss of 17p and 17q was identified in 53 and 29% of informative cases, respectively. p53 nuclear overexpression was detected in 34% of the tumors analyzed. We observed an association between 17p deletions and tumor presentation being more frequent in recurrent and metastatic tumors than primary lesion. p53 nuclear overexpression was associated with tumor grade, size, and more frequently detected in metastatic than primary sarcomas. The 11 intragenic mutations characterized included 10 cases of single base substitution and one single base deletion; 8 were of the missense type and 3 were nonsense. It is concluded that 17p deletions and TP53 mutations are common events in adult soft tissue sarcomas and that due to the trends observed with the cohort of patients analyzed they may become prognostic markers for patients affected with these tumors.

Prognostic implications of p53 nuclear overexpression and high proliferation index of Ki-67 in adult soft-tissue sarcomas.

BACKGROUND: Morphologically similar soft-tissue sarcomas may behave in very different fashions, making it difficult to predict clinical outcomes and to properly design therapeutic interventions. In a preliminary study, we observed that TP53 mutations and nuclear overexpression of p53 protein were frequent events in soft-tissue sarcoma, and we noticed an association between p53-positive phenotype and poor clinical outcome. PURPOSE: We examined the potential clinical relevance of p53 overexpression in adults with soft-tissue sarcomas. We also studied the clinical implications of a high proliferation index. METHODS: A cohort of 174 adults with soft-tissue sarcomas were analyzed using anti-p53 and anti-Ki-67 antibodies and immunohistochemical assays on consecutive fresh frozen tissue samples. RESULTS: We observed a significant association between p53 nuclear overexpression and tumor grade (P = .001) and tumor size (P = .01). Patients displaying a p53-positive phenotype had significantly reduced survival (P = .02). Similarly, a significant difference was observed between high proliferation index and tumor grade (P < .001) and reduced patient survival (P = .03). A high Ki-67 proliferation index was detected in association with p53 nuclear overexpression. CONCLUSIONS: Overexpression of p53 protein and a high proliferation index strongly correlate with poor clinical outcome and reduced survival in patients having soft-tissue sarcomas.

Molecular abnormalities of mdm2 and p53 genes in adult soft tissue sarcomas.

Genetic alterations in the p53 and mdm2 genes have been reported to occur in soft tissue sarcomas. This study was designed to determine the prevalence and potential clinical value of detected molecular abnormalities and altered patterns of expression of mdm2 and p53 genes in adult soft tissue sarcomas. A cohort of 211 soft tissue sarcomas from adults that were both clinically and pathologically well characterized was analyzed. Monoclonal antibodies directed against mdm2 and p53 proteins were used to measure overexpression of these proteins in the nuclei of cells from sections of these tumors. Seventy-six of 207 tumors had abnormally high levels of mdm2 proteins and 56 of 211 tumors overexpressed p53 protein. Twenty-two cases had abnormally high levels of both mdm2 and p53 proteins based upon immunoreactivity with these antibodies. There was a striking statistically significant correlation between the overexpression of p53 and mdm2 proteins in the same tumor and poor survival (P < 0.05) of the patients. A group of 73 soft tissue sarcomas was chosen for analysis using Southern blots, single strand conformation polymorphisms, and direct DNA sequencing to confirm mdm2 gene amplifications and p53 mutations and correlate these with the results of the immunoreactivities. The overexpression of p53 and mdm2 proteins in the nuclei of tumor cells did not always correlate well with gene amplification at the mdm2 locus or mutation at the p53 gene. The possible reasons for these discrepancies are discussed.

A radio-enzymatic method for the estimation of L-leucine-specific radioactivity.

A method is presented for the estimation of L-leucine concentration and radioactivity in biological samples. The sample L-leucine is specifically bound to tRNA and its radioactivity estimated in the presence of either added labelled L-leucine or cold L-leucine (in the same proportion), as well as in the presence of a large excess of cold L-leucine. The latter gave the measurement of non-leucine radioactivity present in the sample. The measurements in the presence and absence of labelled/cold L-leucine allowed the estimation of L-leucine levels and radioactivity by using a simple set of calculations and a standard curve built with known cold L-leucine concentrations in the presence of a fixed known amount of [14C]L-leucine.

Short-term oscillations of aortic core temperature and thermogenic organ blood flow in the rat.

Core and organ temperatures in the rat have been found to be subject to regular periodic oscillations (with a frequency of ca 12 cycles per day, i.e. 143 mu Hz) within set limits, by using a chronic thermocouple implant procedure. These cycles consisted of consecutive warming (the temperature increased steadily) and cooling periods (the temperature decreased regularly). There were sustained temperature differences between several tissues and that of the aortic blood (core temperature), higher for brown adipose tissue, kidney and liver, and lower for muscle, white adipose tissue and skin. Organ blood flows and cardiac output were measured by a radioactive microsphere retention method applied to unanaesthetized rats. Blood flow measurements were done on unaware rats (see Methods), during either the warming or cooling phases. During the warming phases, there was a higher blood flow across the brown adipose tissue, kidney and skin, with a cardiac output about twice that found in the cooling periods. The haemodynamic changes observed, as well as the organ temperature differences observed with respect to the blood, suggest that periodic changes in blood flow are an essential part of the operation of the thermogenic system.

Measurement of rat lung blood flow with labelled microspheres.

A modification of the usual microsphere injection method is presented which is appropriate for rat lung blood flow measurement. The injection of microspheres into the abdominal cava vein, together with the sampling of a reference flow from the right ventricle, allowed to calculate pulmonary blood flows in the same range than the cardiac output. The differences between the two figures (about 30%) are attributed to arterio-venous shunts. The tissue distribution of the microspheres bypassing the lung capillary beds agree with this interpretation.

An enzymatic method for the estimation of L-leucine in rat blood.

A specific enzymatic method for the routine measurement of L-leucine in blood samples is presented. The method uses a commercial preparation of tRNAs and amino acetyl-tRNA synthetases for the specific loading of L-leucine into the tRNA(Leu) present, competing with carrier-free L-[U-14C]leucine. The radioimmunoassay-like plot of radioactivity found in the acid-insoluble (tRNA) fraction was used to determine the amount of unlabelled L-leucine of the samples when compared against a standard curve. The interference of L-isoleucine, L-valine and L-alanine was very low.