The chemotherapeutic drug doxorubicin does not exacerbate p16Ink4a-positive senescent cell accumulation and cardiometabolic disease development in young adult female LDLR-deficient mice.

Cancer survivors who received chemotherapy, such as the anthracycline doxorubicin (DOX), have an increased risk of developing complications later in life, including the development of chronic metabolic diseases. Although the etiology of this increased risk for late metabolic complications in cancer survivors is poorly understood, a causal role of therapy-induced senescent cells has been suggested. To study the role of cellular senescence in chemotherapy-induced metabolic complications, young adult female low-density lipoprotein receptor-deficient (Ldlr-/-)-p16-3MR mice, in which p16Ink4a-positive (p16Ink4a+) senescent cells can be genetically eliminated, were treated with four weekly injections of DOX (2.5 mg/kg) followed by a high-fat high-cholesterol diet for 12 weeks. While DOX treatment induced known short-term effects, such as reduction in body weight, gonadal fat mass, and adipose tissue inflammation, it was not associated with significant long-term effects on glucose homeostasis, hepatic steatosis, or atherosclerosis. We further found no evidence of DOX-induced accumulation of p16Ink4a+-senescent cells at 1 or 12 weeks after DOX treatment. Neither did we observe an effect of elimination of p16Ink4a+-senescent cells on the development of diet-induced cardiometabolic complications in DOX-treated mice. Other markers for senescence were generally also not affected except for an increase in p21 and Cxcl10 in gonadal white adipose tissue long-term after DOX treatment. Together, our study does not support a significant role for p16Ink4a+-senescent cells in the development of diet-induced cardiometabolic disease in young adult DOX-treated female Ldlr-/- mice. These findings illustrate the need of further studies to understand the link between cancer therapy and cardiometabolic disease development in cancer survivors.

Bazi Bushen capsule attenuates cognitive deficits by inhibiting microglia activation and cellular senescence.

CONTEXT: Bazi Bushen capsule (BZBS) has anti-ageing properties and is effective in enhancing memory. OBJECTIVE: To find evidence supporting the mechanisms and biomarkers by which BZBS functions. MATERIALS AND METHODS: Male C57BL/6J mice were randomly divided into five groups: normal, ageing, ß-nicotinamide mononucleotide capsule (NMN), BZBS low-dose (LD-BZ) and BZBS high-dose (HD-BZ). The last four groups were subcutaneously injected with d-galactose (d-gal, 100 mg/kg/d) to induce the ageing process. At the same time, the LD-BZ, HD-BZ and NMN groups were intragastrically injected with BZBS (1 and 2 g/kg/d) and NMN (100 mg/kg/d) for treatment, respectively. After 60 days, the changes in overall ageing status, brain neuron morphology, expression of p16INK4a, proliferating cell nuclear antigen (PCNA), ionized calcium-binding adapter molecule 1 (Iba1), postsynaptic density protein 95 (PSD95), CD11b, Arg1, CD206, Trem2, Ym1 and Fizz1, and the senescence-associated secretory phenotype (SASP) factors were observed. RESULTS: Compared with the mice in the ageing group, the HD-BZ mice exhibited obvious improvements in strength, endurance, motor coordination, cognitive function and neuron injury. The results showed a decrease in p16INK4a, Iba1 and the upregulation of PCNA, PSD95 among brain proteins. The brain mRNA exhibited downregulation of Iba1 (p < 0.001), CD11b (p < 0.001), and upregulation of Arg1 (p < 0.01), CD206 (p < 0.05), Trem2 (p < 0.001), Ym1 (p < 0.01), Fizz1 (p < 0.05) and PSD95 (p < 0.01), as well as improvement of SASP factors. CONCLUSIONS: BZBS improves cognitive deficits via inhibition of cellular senescence and microglia activation. This study provides experimental evidence for the wide application of BZBS in clinical practice for cognitive deficits.

Villosol reverses 5-FU resistance in colorectal cancer by inhibiting the CDKN2A gene regulated TP53-PI3K/Akt signaling axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Patrinia villosa (Juss.) (PV) is the drug of choice in traditional Chinese medicine for the treatment of colorectal cancer (CRC) and has achieved reliable efficacy in clinic. Villosol is the active ingredient in PV. However, the molecular mechanism by which Villosol reverses chemoresistance in CRC remains unclear. AIM OF THE STUDY: Analysis of the molecular mechanism by which Villosol, the active ingredient of PV, reverses CRC/5-FU resistance through modulation of the CDKN2A gene was validated by network pharmacology techniques and experiments. MATERIALS AND METHODS: We identified CDKN2A as a gene associated with 5-FU resistance through gene chip analysis. Next, we conducted a series of functional analyses in cell lines, animal samples, and xenograft models to investigate the role, clinical significance, and abnormal regulatory mechanisms of CDKN2A in 5-FU resistance in CRC. In addition, we screened and obtained a raw ingredient called Villosol, which targets CDKN2A, and investigated its pharmacological effects. RESULTS: Analysis of CRC cells and animal samples showed that the upregulation of CDKN2A expression was strongly associated with 5-FU resistance. CRC cells overexpressing CDKN2A showed reduced sensitivity to 5-FU and enhanced tumor biology in vitro. Inhibition of aberrant activation of CDKN2A enhances the expression of TP53. Mechanistically, overexpression of CDKN2A activates the PI3K/Akt pathway and induces resistance to 5-FU. Villosol inhibited CDKN2A, and CRC/5-FU cells regained sensitivity to 5-FU. Villosol effectively reverses 5-FU resistance through the CDKN2A-TP53-PI3K/Akt axis. CONCLUSION: Changes in CDKN2A gene expression can be used to predict the response of CRC patients to 5-FU therapy. Additionally, inhibiting CDKN2A activation with Villosol may present a new approach to overcoming 5-FU resistance in clinical settings.

Pharmacological Depletion of Microglia Leads to a Dose-Dependent Reduction in Inflammation and Senescence in the Aged Murine Brain.

Chronic macrophage activation was implicated as one of the main culprits for chronical, low-grade inflammation which significantly contributes to development of age-related diseases. Microglia as the brain macrophages have been recently implicated as key players in neuroinflammation and neurodegeneration in the aged brain. Microglial cell functions are indispensable in early development, however, activation or senescence of microglia in aging cells may be detrimental. Depletion of microglia using genetical or pharmacological approaches leads to opposite results regarding effects on brain cognition. In this study we pharmacologically depleted microglia using orally delivered low and high doses of the CSF1R inhibitor PLX5622 and assessed the expression levels of known inflammation markers (TNF-α, IL1-ß, IL-6, IL-10), glia markers (Iba-1 and Gfap) and specific senescence marker p16Ink4a in the aged murine brain. Our results indicate that treatment with low and high doses of PLX5622 leads to a dose-dependent depletion of microglial cells with similar levels in young and aged mice. We also show that treatment with low and high PLX5622 differentially affected cytokine levels in young and old brains. By using low doses we could achieve reduction in inflammation circumventing the astrocyte activation. Removal of microglia cells led to decreased expression of the senescence marker p16Ink4a in the aged brain, indicating a relevant contribution of these cells to the expression of this marker and their senescent status in the healthy aging brain. Our results indicate that increased and detrimental brain inflammation in aged murine brain can be impaired by selectively reducing the microglial cell population.

Bioactive Oligopeptides from Ginseng (Panax ginseng Meyer) Suppress Oxidative Stress-Induced Senescence in Fibroblasts via NAD+/SIRT1/PGC-1α Signaling Pathway.

The physicochemical properties and multiple bioactive effects of ginseng oligopeptides (GOPs), plant-derived small molecule bioactive peptides, suggest a positive influence on health span and longevity. Given this, cellular senescence is the initiating factor and key mechanism of aging in the organism, and thus the current study sought to explore the effects of GOPs on H2O2-induced cellular senescence and its potential mechanisms. Senescence was induced in mouse embryonic fibroblasts NIH/3T3 by 4 h of exposure to 200 µM H2O2 and confirmed using CCK-8 assay and Western blot analyses of p16INK4A and p21Waf1/Cip1 after 24 h of growth medium administration with or without GOPs supplementation (25, 50, and 100 µg/mL). We found that GOPs delayed oxidative stress-induced NIH/3T3 senescence by inhibiting the G1 phase arrest, increasing DNA synthesis in the S phase, decreasing the relative protein expression of p16INK4A and p21Waf1/Cip1, promoting cell viability, protecting DNA, and enhancing telomerase (TE) activity. Further investigation revealed that the increase in antioxidative capacity and anti-inflammation capacity might form the basis for the retarding of the senescence effects of GOPs. Furthermore, GOPs supplementation significantly improved mitochondrial function and mitochondrial biogenesis via the NAD+/SIRT1/PGC-1𝛼 pathway. These findings indicate that GOPs may have a positive effect on health span and lifespan extension via combating cellular senescence, oxidative stress, and inflammation, as well as modulating longevity regulating pathway NAD+/SIRT1/PGC-1𝛼.

Cellular Senescence in Diabetes Mellitus: Distinct Senotherapeutic Strategies for Adipose Tissue and Pancreatic ß Cells.

Increased insulin resistance and impaired insulin secretion are significant characteristics manifested by patients with type 2 diabetes mellitus (T2DM). The degree and extent of these two features in T2DM vary among races and individuals. Insulin resistance is accelerated by obesity and is accompanied by accumulation of dysfunctional adipose tissues. In addition, dysfunction of pancreatic ß-cells impairs insulin secretion. T2DM is significantly affected by aging, as the ß-cell mass diminishes with age. Moreover, both obesity and hyperglycemia-related metabolic changes in developing diabetes are associated with accumulation of senescent cells in multiple organs, that is, organismal aging. Cellular senescence is defined as a state of irreversible cell cycle arrest with concomitant functional decline. It is caused by telomere shortening or senescence-inducing stress. Senescent cells secrete proinflammatory cytokines and chemokines, which is designated as the senescence-associated secretory phenotype (SASP), and this has a negative impact on adipose tissues and pancreatic ß-cells. Recent advances in aging research have suggested that senolysis, the removal of senescent cells, can be a promising therapeutic approach to prevent or improve aging-related diseases, including diabetes. The attenuation of a SASP may be beneficial, although the pathophysiological involvement of cellular senescence in diabetes is not fully understood. In the clinical application of senotherapy, tissue-context-dependent senescent cells are increasingly being recognized as an issue to be solved. Recent studies have observed highly heterogenic and complex senescent cell populations that serve distinct roles among tissues, various stages of disease, and different ages. For example, in high-fat-diet induced diabetes with obesity, mouse adipose tissues display accumulation of p21Cip1-highly-expressing (p21high) cells in the early stage, followed by increases in both p21high and p16INK4a-highly-expressing (p16high) cells in the late stage. Interestingly, elimination of p21high cells in visceral adipose tissue can prevent or improve insulin resistance in mice with obesity, while p16high cell clearance is less effective in alleviating insulin resistance. Importantly, in immune-deficient mice transplanted with fat from obese patients, dasatinib plus quercetin, a senolytic cocktail that reduces the number of both p21high and p16high cells, improves both glucose tolerance and insulin resistance. On the other hand, in pancreatic ß cells, p16high cells become increasingly predominant with age and development of diabetes. Consistently, elimination of p16high cells in mice improves both glucose tolerance and glucose-induced insulin secretion. Moreover, a senolytic compound, the anti-Bcl-2 inhibitor ABT263 reduces p16INK4a expression in islets and restores glucose tolerance in mice when combined with insulin receptor antagonist S961 treatment. In addition, efficacy of senotherapy in targeting mouse pancreatic ß cells has been validated not only in T2DM, but also in type 1 diabetes mellitus. Indeed, in non-obese diabetic mice, treatment with anti-Bcl-2 inhibitors, such as ABT199, eliminates senescent pancreatic ß cells, resulting in prevention of diabetes mellitus. These findings clearly indicate that features of diabetes are partly determined by which or where senescent cells reside in vivo, as adipose tissues and pancreatic ß cells are responsible for insulin resistance and insulin secretion, respectively. In this review, we summarize recent advances in understanding cellular senescence in adipose tissues and pancreatic ß cells in diabetes. We review the different potential molecular targets and distinctive senotherapeutic strategies in adipose tissues and pancreatic ß cells. We propose the novel concept of a dual-target tailored approach in senotherapy against diabetes.

[Effect of metabolizable peptide p16INK4a on short-lived human tumor cultures].

The study was concerned with antitumor action of internalized peptide incorporating a fragment of p161INK4a using a model of short-lived human tumor cultures sampled from resected material. Renal cancer sample showed the greatest therapeutic interval.

Sexually dimorphic leanness and hypermobility in p16Ink4a/CDKN2A-deficient mice coincides with phenotypic changes in the cerebellum.

p16Ink4a/CDKN2A is a tumor suppressor that critically regulates the cell cycle. Indeed, p16Ink4a deficiency promotes tumor formation in various tissues. We now report that p16Ink4a deficiency in female mice, but not male mice, induces leanness especially in old age, as indicated by lower body weight and smaller white adipose tissue, although other major organs are unaffected. Unexpectedly, the integrity, number, and sizes of adipocytes in white adipose tissue were unaffected, as was macrophage infiltration. Hence, hypermobility appeared to be accountable for the phenotype, since food consumption was not altered. Histological analysis of the cerebellum and deep cerebellar nuclei, a vital sensorimotor control center, revealed increased proliferation of neuronal cells and improved cerebellum integrity. Expression of estrogen receptor ß (ERß) and PCNA also increased in deep cerebellar nuclei, implying crosstalk between p16Ink4a and ERß. Furthermore, p16Ink4a deficiency expands LC3B+ cells and GFAP+ astrocytes in response to estrogen. Collectively, the data suggest that loss of p16INK4a induces sexually dimorphic leanness in female mice, which appears to be due to protection against cerebellar senescence by promoting neuronal proliferation and homeostasis via ERß.

Inhibition of both mesothelioma cell growth and Cdk4 activity following treatment with a TATp16INK4a peptide.

UNLABELLED: Disruption of the 9p21 locus is common in mesothelioma and leads to loss of both the p16INK4a and the p14ARF gene products. This study tested the hypothesis that reexpression of p16INK4a carried out using the TAT delivery system that carries the protein transduction domain of the HIV TAT will result in mesothelioma cell death. MATERIALS AND METHODS: A synthetic TATp16INK4a peptide and a charge matched control were transduced into mesothelioma cells in vitro and in vivo. Cells were assayed for Cdk4 inhibition, cell cycle arrest, and cell death. RESULTS: Treatment of mesothelioma cells with TATp16INK4a for 48 hours resulted in cell death. Apoptosis and G1 cell cycle arrest was also observed. Following transduction of cells with TATp16INK4a there was complete but transient hypophosphorylation of pRb. Similar effects were observed in mesothelioma xenografts. CONCLUSION: Therapeutic strategies which introduce either TATp16INK4a peptide, or small molecule mimetic, could be an effective strategy for mesothelioma treatment.

CAK-independent activation of CDK6 by a viral cyclin.

In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16(INK4a). Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16(INK4a) sensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6(T177A) together with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK.

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell.

To investigate the effect of cell cycle inhibitor p14ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results revealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10-12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-beta-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.

[Cytotoxicity of chimera peptides incorporating sequences of cyclin kinases inhibitors].

The study is concerned with proapoptotic properties of chimera peptides which incorporate sequences of inhibitors of cyclin kinases p161NK4a and p21CIP/WAF1 as well as internalized sequences (Antp and tat). Sequences of the p16 type appeared to be more cytotoxic than the p21 one. Cytotoxic effect proved dependent on orientation with respect to the C or N terminal point of a polypeptide chain rather than on chimera sequence extent. Although p16 endogenous synthesis did not influence chimera peptide levels, apoptosis did not take place in certain cellular lines. Due to the rather unsophisticated nature of such synthesis, it might be used in designing individually-tailored chemotherapeutic drugs.

Suppression of U937 human monocytic leukemia cell growth by dideoxypetrosynol A, a polyacetylene from the sponge Petrosia sp., via induction of Cdk inhibitor p16 and down-regulation of pRB phosphorylation.

Dideoxypetrosynol A, a polyacetylene from the marine sponge Petrosia sp., is known to exhibit significant selective cytotoxic activity against a small panel of human tumor cell lines, the mechanisms of which however, are poorly understood. The aim of the present study was to further elucidate the possible mechanisms by which dideoxypetrosynol A exerts its anti-proliferative action in cultured human monocytic leukemia U937 cells. We observed that the proliferation-inhibitory effect of dideoxypetrosynol A was due to the induction of G1 arrest in the cell cycle, the effects of which were associated with up-regulation of cyclin D1 and down-regulation of cyclin E, in a concentration-dependent manner without any change in cyclin-dependent-kinases (Cdks) expression. Dideoxypetrosynol A markedly induced the levels of Cdk inhibitor p16/INK4a expression. Furthermore, down-regulation of phosphorylation of retinoblastoma protein (pRB) by this compound was associated with enhanced binding of pRB and transcription factor E2F-1. Overall, our results demonstrate a combined mechanism involving the inhibition of pRB phosphorylation and induction of p16 as targets for dideoxypetrosynol A, may explain some of its anti-cancer effects.

Crystal structure of a viral cyclin, a positive regulator of cyclin-dependent kinase 6.

BACKGROUND: Cyclin-dependent kinases (CDKs) have a central role in cell-cycle control and are activated by complex formation with positive regulatory proteins called cyclins and by phosphorylation. The overexpression and mutation of cyclins and CDKs has been associated with tumorigenesis and oncogenesis. A virus-encoded cyclin (v-cyclin) from herpesvirus saimiri has been shown to exhibit highest sequence homology to type D cyclins and specifically activates CDK6 of host cells to a very high degree. RESULTS: We have determined the first X-ray structure of a v-cyclin to 3.0 A resolution. The structure of the core domains is very similar to those of cyclin A and cyclin H from human cells. To understand the structural basis for the v-cyclin specificity for CDK6 and the insensitivity of the complex to inhibitors of the p21 and INK4 families, a v-cyclin-CDK2 model was built on the basis of the known structures of human cyclin A in complex with CDK2 and the CDK inhibitor p27(Kip1). CONCLUSIONS: Although many critical interactions between cyclin A and CDK2 would be conserved in a v-cyclin-CDK2 complex, some appear sterically or electrostatically unfavorable due to shifts in the backbone conformation or sidechain differences and may contribute to v-cyclin selectivity for CDK6. The insensitivity of v-cyclin-CDK6 complexes to inhibitors of the p21 family is probably due to structural changes in v-cyclin that lead to a flatter surface area offering fewer potential contacts with the protein inhibitor. In addition, sequence changes in v-cyclin eliminate hydrogen-bonding partners for atoms of the p27(Kip1) inhibitor. This structure provides the first model for interactions between v-cyclins and host cell-cycle proteins; these interactions may be important for virus survival as well as oncogenic transformation of host cells.

Cyclin D-dependent kinases, INK4 inhibitors and cancer.

The Cyclin D-Cdk4,6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The components of this pathway are gene families with a high level of structural and functional redundancy and are expressed in an overlapping fashion in most tissues and cell types. Using classical transgenic technology as well as gene-targeting in ES cells, a series of mouse models have been developed to study the in vivo function of individual components of this pathway in both normal homeostasis and tumor development. These models have proven to be useful to define specific as well as redundant roles among members of these cell cycle regulatory gene families. This pathway is deregulated in the vast majority of human tumors by genetic and epigenetic alterations that target at least some of its key members such as Cyclin D1, Cdk4, INK4a and INK4b, pRb etc. As a consequence, some of these molecules are currently being considered as targets for cancer therapy, and several novel molecules, such as Cdk inhibitors, are under development as potential anti-cancer drugs.

Suppression of tumorigenesis and induction of p15(ink4b) by Smad4/DPC4 in human pancreatic cancer cells.

PURPOSE: The tumor suppressor gene Smad4/DPC4, a key transcription factorin transforming growth factor beta (TGF-beta) signaling cascades,is inactivated in 50% of pancreatic adenocarcinomas. We seek to determine the role of Smad4/DPC4 in the suppression of tumor cell growth and in the regulation of TGF-beta-mediated expression of cell-cycle regulatory genes p15(ink4b) and p21(waf1). EXPERIMENTAL DESIGN: Smad4/DPC4 is overexpressed by adenoviral infection in CFPac-1 pancreatic cancer cells, in which the Smad4/DPC4 is homozygously deleted, and in Capan-1 pancreatic cancer cells, in which Smad4/DPC4 is not expressed. Expression of the TGF-beta downstream target gene p21(waf1), regulation of the p15(ink4b) promoter, anchorage-independent growth, and tumorigenesis were examined. RESULTS: We demonstrate that expression of Smad4/DPC4 in Capan-1 cells reduced anchorage-independent growth by more than 50%, and inhibited xenograft tumor growth. However, overexpression of Smad4/DPC4 did not inhibit CFPac-1 cell growth. Interestingly, Smad4/DPC4 induced expression of p15(ink4b), p21(waf1), and TGF-beta-responsive reporter gene in Capan-1 but not in CFPac-1 cells. Furthermore, we found a previously unidentified Smad4 binding element (SBE) located in the region between -356 and -329 bp of the p15(ink4b) promoter. The p15(ink4b) promoter reporter gene assays revealed that Smad4-dependent transcriptional activation is mediated by this SBE, which indicates that p15(ink4b) is one of the downstream target genes regulated by Smad/DPC4. CONCLUSION: These results explain the role of Smad4/DPC4 in TGF-beta-mediated inhibition of cell proliferation in vitro and in vivo. Moreover, these results suggest that Smad4/DPC4-mediated tumor suppression and induction of TGF-beta-regulated cell-cycle-inhibitory genes may depend on additional factors that are absent in CFPac-1 cells.

Effect of exogenous p16ink4a and hRb1 genes on cell cycle regulation of osteosarcoma cell.

To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRb1 genes, pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIRES-hRb1 plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium. mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western-Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectively in vitro. It was demonstrated with subG1 flow cytometric analysis and MTT method that p16ink4a and hRb1 genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone (P < 0.01). Coexpression of exogenous p16ink4a with hRb1 broke the regulatory feedback loop of p16ink4a-cyclinD1 /CDK-hRb1 and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRb1 did alone in vitro.

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1.

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7-Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4(+) and CD8(+) lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.

Enhanced TGFalpha-EGFR expression and P53 gene alterations contributes to gastric tumors aggressiveness.

We determined whether alterations in the expression of p53, p16(INK4) and p21(WAF1/CIP1) influence the invasiveness of a subset of gastric adenocarcinomas co-expressing TGFalpha and EGFR. Immunopositivity for TGFalpha-EGFR (26%) was observed in both early and advanced adenocarcinomas, and 88% of these showed immunoreactivity for p53. SSCP analysis revealed that in 81% of these tumors the p53 gene was mutated in exons 5-8. The intensity of p53 immunoreactivity was significantly higher (P < 0.013) in deeply invasive tumors. p16(INK4) and p21(WAF1/CIP1) immunoreactivity was detected in 93 and 76% of the samples co-expressing TGFalpha-EGFR but the levels were not correlated with those of p53 and other clinico-pathological parameters. We conclude that gastric adenocarcinomas potentially dependent upon the TGFalpha-EGFR autocrine loop for growing exhibit increased aggressiveness in the presence of aberrant p53.