The terminal differentiation of C2C12 skeletal muscle cells involves the activation of unique sets of genes and an irreversible withdrawal from the cell cycle. This process is associated with a decrease in cdk2 activity in cell extracts. The decrease in cdk2 activity correlates with diminished levels of cdk2 and cyclin A and with a marked induction of the p21 cyclin-dependent kinase (cdk) inhibitor. The upregulation of p21 occurred at the levels of mRNA and protein, and p21 formed a complex with the cyclin kinases in myotubes. Further, the immunodepletion of p21 from myotube extracts neutralized the heat-stable cdk2 inhibitory activity that was induced upon myogenic differentiation. The levels of p21 mRNA, protein, and activity remained constant in myotubes when they were reexposed to mitogen-rich growth medium, indicating that permanent changes in the cell's genetic program contribute to its sustained expression following terminal differentiation. Indeed, 10T1/2 fibroblasts transformed with the myogenic factor MyoD, but not the parental multipotent cells, upregulated p21 transcript levels when induced to differentiate by serum withdrawal, demonstrating that the upregulation is an integral feature of myogenic commitment and differentiation. The functional consequences of this upregulation were indicated by ectopically expressing p21 in myoblasts; this was sufficient for cell cycle arrest in mitogen-rich growth medium. The induction and sustained expression of p21 appears to be a contributory mechanism by which myocytes irreversibly exit the cell cycle upon terminal differentiation.

PD-L1 immunohistochemistry correlates only moderately with patient survival and response to PD-(L)1 treatment. Heterogeneity of tumor PD-L1 expression might limit the predictive value of small biopsies. Here we show that tumor PD-L1 and PD-1 expression can be quantified non-invasively using PET-CT in patients with non-small-cell lung cancer. Whole body PD-(L)1 PET-CT reveals significant tumor tracer uptake heterogeneity both between patients, as well as within patients between different tumor lesions.

The identification of antigenic peptides presented on the tumor cell surface by HLA class I molecules and recognized by tumor-specific cytotoxic T lymphocytes may lead to a peptide vaccine capable of inducing protective cellular immunity. We demonstrate that both HLA-A2-restricted breast and ovarian tumor-specific cytotoxic T lymphocytes recognize shared (3) and MART (melanoma antigen recognized by T cells) (4) in melanoma. The discovery of these genes and peptides substantiates the existence of such antigen systems, but unfortunately, melanoma accounts for only 3% of all malignancies and 1% of all cancer deaths (5).HLA-A2 is expressed in approximately 50% of Caucasians (6) and has been demonstrated to play a critical role in antigen presentation of both viral antigens (7) and tumor antigens from a variety of cancers (8-10). HER2/neu is a 185-kDa transmembrane glycoprotein with tyrosine kinase activity and extensive homology to the epidermal growth factor receptor (11). HER2/neu is ubiquitously expressed in many tumors and known to be overexpressed in approximately 30-40% of all ovarian and breast cancers (12).We have shown that tumor-specific CTLs can be demonstrated in the tumor-infiltrating lymphocytes (TILs) isolated from ovarian cancers (13). We have also found that HLA-A2 presents TAA in this disease (14). The level of expression of the HER2/neu oncogene in ovarian cancer positively correlates with recognition by HLA-A2+ ovarian tumor-specific CTLs, and HLA-A2+ melanoma cells transfected with the HER2/neu gene become sensitive to ovarian cancer-specific CTLs (15). Therefore, HLA-A2-presented, HER2/neuderived antigenic peptides may be recognized by ovarian cancer-specific CTLs and, potentially, by breast cancerspecific CTLs as well.In this study, we have searched the HER2/neu sequence (16) for HLA-A2-binding peptides (17), and we have found that both ovarian and breast cancer-specific CTLs recognize a nine-amino acid peptide from the transmembrane portion of the HER2/neu protein (GP2; amino acids 654-662). This peptide is widely expressed in HER2/neu+ tumors and is capable of inducing HLA-A2-restricted, tumor-specific CTL populations in vitro. METHODSGeneration of Tumor-Specific CTLs. Tumor-specific CTLs were generated from fresh tumor specimens obtained through the Departments of Surgery, Gynecologic Oncology, and Pathology at Brigham and Women's Hospital (BWH) and Beth Israel Hospital in Boston, under approval of the Institutional Review Boards. Solid tumor specimens were processed as previously described (14). Briefly, specimens were minced manually and enzymatically digested, and the lymphocytes and tumor cells were separated by centrifugation over discontinuous Ficoll (Organon Teknika-Cappel) gradients. TILs were suspended in RPMI-1640 medium (BioWhittaker) supplemented with 10% fetal calf serum (BioWhittaker) and antibiotics. Cultures were suspended at 5 x 105 cells per ml on solid-phase, anti-CD3 polystyrene plates (Orthoclone OKT3, Ortho Pharmaceuticals) for 48 h. T cells were then mainta...

Intracellular ␤-amyloid 42 (A␤42) accumulation is increasingly recognized as an early event in the pathogenesis of Alzheimer's disease (AD). We have developed a doxycycline-inducible adenoviral-based system that directs intracellular A␤42 expression and accumulation into the endoplasmic reticulum of primary neuronal cultures in a regulated manner. A␤42 exhibited a perinuclear distribution in cell bodies and an association with vesicular compartments. Virally expressed intracellular A␤42 was toxic to neuronal cultures 24 hr after induction in a dose-dependent manner. A␤42 expression prompted the rapid induction of stress-inducible Hsp70 protein in neurons, and virally mediated Hsp70 overexpression rescued neurons from the toxic effects of intracellular A␤ accumulation. Together, these results implicate the cellular stress response as a possible modulator of A␤-induced toxicity in neuronal cultures.

gax, a diverged homeobox gene expressed in vascular smooth muscle cells (VSMCs), is down-regulated in vitro by mitogen stimulation and in vivo in response to vascular injury that leads to cellular proliferation. Recombinant Gax protein microinjected into VSMCs and fibroblasts inhibited the mitogen-induced entry into S-phase when introduced either during quiescence or early stages of G1. Overexpression of gax with a replication-defective adenovirus vector resulted in Go/G 1 cell cycle arrest of VSMCs and fibroblasts. The gax-induced growth inhibition correlated with a p53-independent up-regulation of the cyclin-dependent kinase inhibitor p21. Gax overexpression also led to an association of p21 with cdk2 complexes and a decrease in cdk2 activity. Fibroblasts deficient in p21 were not susceptible to a reduction in cdk2 activity or growth inhibition by gax overexpression. Localized delivery of the virus to denuded rat carotid arteries significantly reduced neointima formation and luminal narrowing. These data indicate that gax overexpression can inhibit cell proliferation in a p21-dependent manner and can modulate injury-induced changes in vessel wall morphology that result from excessive cellular proliferation.

The programmed death protein (PD-1) and its ligand (PD-L1) play critical roles in a checkpoint pathway cancer cells exploit to evade the immune system. A same-day PET imaging agent for measuring PD-L1 status in primary and metastatic lesions could be important for optimizing drug therapy. Herein, we have evaluated the tumor targeting of an anti-PD-L1 adnectin after F-fluorine labeling. An anti-PD-L1 adnectin was labeled with F in 2 steps. This synthesis featured fluorination of a novel prosthetic group, followed by a copper-free click conjugation to a modified adnectin to generateF-BMS-986192. F-BMS-986192 was evaluated in tumors using in vitro autoradiography and PET with mice bearing bilateral PD-L1-negative (PD-L1(-)) and PD-L1-positive (PD-L1(+)) subcutaneous tumors.F-BMS-986192 was evaluated for distribution, binding, and radiation dosimetry in a healthy cynomolgus monkey. F-BMS-986192 bound to human and cynomolgus PD-L1 with a dissociation constant of less than 35 pM, as measured by surface plasmon resonance. This adnectin was labeled withF to yield a PET radioligand for assessing PD-L1 expression in vivo. F-BMS-986192 bound to tumor tissues as a function of PD-L1 expression determined by immunohistochemistry. Radioligand binding was blocked in a dose-dependent manner. In vivo PET imaging clearly visualized PD-L1 expression in mice implanted with PD-L1(+), L2987 xenograft tumors. Two hours after dosing, a 3.5-fold-higher uptake (2.41 ± 0.29 vs. 0.82 ± 0.11 percentage injected dose per gram, < 0.0001) was observed in L2987 than in control HT-29 (PD-L1(-)) tumors. Coadministration of 3 mg/kg ADX_5322_A02 anti-PD-L1 adnectin reduced tumor uptake at 2 h after injection by approximately 70%, whereas HT-29 uptake remained unchanged, demonstrating PD-L1-specific binding. Biodistribution in a nonhuman primate showed binding in the PD-L1-rich spleen, with rapid blood clearance through the kidneys and bladder. Binding in the PD-L1(+) spleen was reduced by coadministration of BMS-986192. Dosimetry estimates indicate that the kidney is the dose-limiting organ, with an estimated human absorbed dose of 2.20E-01 mSv/MBq. F-BMS-986192 demonstrated the feasibility of noninvasively imaging the PD-L1 status of tumors by small-animal PET studies. Clinical studies withF-BMS-986192 are under way to measure PD-L1 expression in human tumors.

A poptotic death of vascular cells is a prominent feature of blood vessel remodeling that occurs during normal development and fibroproliferative disorders of the vessel wall. This review summarizes a large number of studies that have provided evidence for apoptotic cell death in the vasculature. We also describe reports that shed light on the molecular mechanisms that may control this process. Finally, we highlight the relatively small number of studies that suggest a function for vascular cell apoptosis in controlling the morphology and cellular composition of the blood vessel wall.A number of studies have demonstrated apoptotic death of vascular cells in vessels that remodel postnatally. Regionalized apoptosis has been found in vascular smooth muscle cells (VSMCs) during the regression and closure of the human ductus arteriosus before birth 1 and in VSMCs and endothelial cells of the arteries and veins of the umbilical cord, which are subject to dramatic hemodynamic changes at birth. 2 Evidence of VSMC apoptosis in the human neonate has also been found at the branch points of the great arteries arising from the aortic arch when they are exposed to a disturbed blood flow, whereas VSMC apoptosis was not observed in the aorta when a normal flow pattern is maintained. 2 Finally, VSMC apoptosis has been observed during the remodeling of the abdominal aorta in lambs, resulting from the large decrease in blood flow that occurs after the loss of the placenta at birth. 3 Vascular cell apoptosis during neonatal vascular remodeling appears to be triggered by decreased flow or by perturbation of flow at branch points that results when the organism switches oxygen exchange from the placenta to the lungs. Cell loss occurring upon flow-induced vessel remodeling has been directly demonstrated by inducing changes in flow through the carotid arteries of immature rabbits. 4 In that experimental system, ligation of the left external carotid artery results in a marked reduction in blood flow through the common carotid artery, and this reduction correlates with a large increase in endothelial cell and VSMC apoptosis. Presumably, vascular cell apoptosis contributes to an adaptive process that allows the vessel to permanently constrict to manage the decrease in flow.Changes in flow affect wall tension and cell matrix interactions, and it may be these factors that alter the survival characteristics of vascular cells. Consistent with this notion, VSMC apoptosis occurs when wall tension is diminished, 5 or enhanced, 6 or when the expression of matrix components 7 or matrix metalloproteinases 8 is altered. Although changes in the proapoptotic proteins Bax and Bcl-X S are associated with flowinduced vascular remodeling, 2 relatively little is known about the molecular mechanisms that regulate vascular cell viability under these circumstances. Furthermore, although it makes intuitive sense that cellular elimination would be required for negative remodeling of a vessel, causal data in support of this hypothesis have not yet been provided.Ap...

Sonic hedgehog (Shh) is a morphogen regulating crucial epithelial-mesenchymal interactions during embryonic development, but its signaling pathway is considered generally silent in post-natal life. In this study, we demonstrate that Shh is de novo expressed after injury and during regeneration of the adult skeletal muscle. Shh expression is followed by significant upregulation of its receptor and target gene Ptc1 in injured and regenerating muscles. The reactivation of the Shh signaling pathway has an important regulatory role on injury-induced angiogenesis, as inhibition of Shh function results in impaired upregulation of prototypical angiogenic agents, such as vascular endothelial growth factor (VEGF) and stromal-derived factor (SDF)-1alpha, decreased muscle blood flow, and reduced capillary density after injury. In addition, Shh reactivation plays a regulatory role on myogenesis, as its inhibition impairs the activation of the myogenic regulatory factors Myf-5 and MyoD, decreases the upregulation of insulin-like growth factor (IGF)-1, and reduces the number of myogenic satellite cells at injured site. Finally, Shh inhibition results in muscle fibrosis, increased inflammatory reaction, and compromised motor functional recovery after injury. These data demonstrate that the Shh pathway is functionally important for adult skeletal muscle regeneration and displays pleiotropic angiogenic and myogenic potentials in post-natal life. These findings might constitute the foundation for new therapeutic approaches for muscular diseases in humans.