Differentiation between neurofibromas and malignant peripheral nerve sheath tumors in neurofibromatosis 1 evaluated by MRI

Purpose  The imaging discrimination between neurofibroma (NF) and malignant peripheral nerve sheath tumor (MPNST) is clinically very important. The purpose of this study is to define the criteria for the differential diagnosis between NF and MPNST on MRI in neurofibromatosis 1 (NF1). Methods  A total of 37 patients with NF1, 18 NFs and 19 MPNSTs were evaluated by MRI at 1.5 T. Magnetic resonance imaging (MRI) findings were compared using univariate and multivariate analyses. Results  The MRI findings characteristic of MPNST (p < 0.05) were an irregular tumor shape (15/19 in MPNST vs. 5/18 in NF), unclear margin (13/19 in MPNST vs. 6/18 in NF), intra-tumoral lobulation (12/19 in MPNST vs. 3/18 in NF), presence of high signal-intensity area on T1-weighted images (T1WI) (12/19 in MPNST vs. 1/18 in NF), no target sign (0/19 in MPNST vs. 12/18 in NF), inhomogeneous enhancement on contract-enhanced T1WI (17/18 in MPNST vs. 9/16 in NF) and a lower rate of enhanced area (54% in MPNST vs. 87% in NF) were critical indicators to differentiate MPNST from NF. A multivariate analysis showed that intra-tumoral lobulation and the presence of a high signal-intensity area on T1WI were considered to be diagnostic indicators of MPNST. The sensitivity and specificity for these two items were 63.2, 83.3, 63.2 and 87.5%, respectively. Conclusion  MRI shows features which were helpful for differentiating MPNST from NF.     

TGF-beta signaling engages an ATM-CHK2-p53-independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells

Oncogene-induced senescence (OIS), the proliferative arrest engaged in response to persistent oncogene activation, serves as an important tumor-suppressive barrier. We show here that finite lifespan human mammary epithelial cells (HMEC) undergo a p16/RB- and p53-independent OIS in response to oncogenic RAS that requires TGF-β signaling. Suppression of TGF-β signaling by expression of a dominant-negative TGF-β type II receptor, use of a TGF-β type I receptor inhibitor, or ectopic expression of MYC permitted continued proliferation upon RAS expression. Surprisingly, unlike fibroblasts, shRNA-mediated knockdown of ATM or CHK2 was unable to prevent RAS-mediated OIS, arguing that the DNA damage response is not required for OIS in HMEC. Abrogation of TGF-β signaling not only allowed HMEC lacking p53 to tolerate oncogenic RAS but also conferred the capacity for anchorage-independent growth. Thus, the OIS engaged after dysregulated RAS expression provides an early barrier to malignant progression and is mediated by TGF-β receptor activation in HMEC. Understanding the mechanisms that initiate and maintain OIS in epithelial cells may provide a foundation for future therapies aimed at reengaging this proliferative barrier as a cancer therapy.     

The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization

PTEN is a tumor suppressor frequently mutated in cancer. Recent reports implicated Nedd4-1 as the E3 ubiquitin ligase for PTEN that regulates its stability and nuclear localization. We tested the physiological role of Nedd4-1 as a PTEN regulator by using cells and tissues derived from two independently generated strains of mice with their Nedd4-1 gene disrupted. PTEN stability and ubiquitination were indistinguishable between the wild-type and Nedd4-1-deficient cells, and an interaction between the two proteins could not be detected. Moreover, PTEN subcellular distribution, showing prominent cytoplasmic and nuclear staining, was independent of Nedd4-1 presence. Finally, activation of PKB/Akt, a major downstream target of cytoplasmic PTEN activity, and the ability of PTEN to transactivate the Rad51 promoter, a measure of its nuclear function, were unaffected by the loss of Nedd4-1. Taken together, our results fail to support a role for Nedd4-1 as the E3 ligase regulating PTEN stability and subcellular localization.     

Nanoparticles enhance brain delivery of blood-brain barrier-impermeable probes for in vivo optical and magnetic resonance imaging

Several imaging modalities are suitable for in vivo molecular neuroimaging, but the blood-brain barrier (BBB) limits their utility by preventing brain delivery of most targeted molecular probes. We prepared biodegradable nanocarrier systems made up of poly(n-butyl cyanoacrylate) dextran polymers coated with polysorbate 80 (PBCA nanoparticles) to deliver BBB-impermeable molecular imaging probes into the brain for targeted molecular neuroimaging. We demonstrate that PBCA nanoparticles allow in vivo targeting of BBB-impermeable contrast agents and staining reagents for electron microscopy, optical imaging (multiphoton), and whole brain magnetic resonance imaging (MRI), facilitating molecular studies ranging from individual synapses to the entire brain. PBCA nanoparticles can deliver BBB-impermeable targeted fluorophores of a wide range of sizes: from 500-Da targeted polar molecules to 150,000-Da tagged immunoglobulins into the brain of living mice. The utility of this approach is demonstrated by (i) development of a "Nissl stain" contrast agent for cellular imaging, (ii) visualization of amyloid plaques in vivo in a mouse model of Alzheimer's disease using (traditionally) non-BBB-permeable reagents that detect plaques, and (iii) delivery of gadolinium-based contrast agents into the brain of mice for in vivo whole brain MRI. Four-dimensional real-time two-photon and MR imaging reveal that brain penetration of PBCA nanoparticles occurs rapidly with a time constant of ～18 min. PBCA nanoparticles do not induce nonspecific BBB disruption, but collaborate with plasma apolipoprotein E to facilitate BBB crossing. Collectively, these findings highlight the potential of using biodegradable nanocarrier systems to deliver BBB-impermeable targeted molecular probes into the brain for diagnostic neuroimaging.     

Cooperative interactions of BRAFV600E kinase and CDKN2A locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy

Although malignant astrocytomas are a leading cause of cancer-related death in children, rational therapeutic strategies are lacking. We previously identified activating mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) (BRAF(T1799A) encoding BRAF(V600E)) in association with homozygous cyclin-dependent kinase inhibitor 2A (CDKN2A, encoding p14ARF and p16Ink4a) deletions in pediatric infiltrative astrocytomas. Here we report that BRAF(V600E) expression in neural progenitors (NPs) is insufficient for tumorigenesis and increases NP cellular differentiation as well as apoptosis. In contrast, astrocytomas are readily generated from NPs with additional Ink4a-Arf deletion. The BRAF(V600E) inhibitor PLX4720 significantly increased survival of mice after intracranial transplant of genetically relevant murine or human astrocytoma cells. Moreover, combination therapy using PLX4720 plus the Cyclin-dependent kinase (CDK) 4/6-specific inhibitor PD0332991 further extended survival relative to either monotherapy. Our findings indicate a rational therapeutic strategy for treating a subset of pediatric astrocytomas with BRAF(V600E) mutation and CDKN2A deficiency.     

64Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific ⁶⁴Cu-monoclonal antibody (mAb)–TCR complex enables a stable labeling of T cells. The TCR–mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied ⁶⁴Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.Noninvasive in vivo imaging is an emerging method that enables the examination of T-cell migration kinetics, homing patterns, and the sites of T-cell proliferation and activation. These data are needed for a better understanding of the T-cell response during autoimmune diseases, allergies, infections, and cancer (1). Furthermore, this method reveals the basic mechanisms required for the understanding of T-cell-based immunotherapies and the development and optimization of personalized therapies (2). CD4⁺ IFN-γ–producing T-helper cells (TH1) and IL-4–producing T-helper cells (TH2) are key players in organ-specific autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, bronchial asthma, and insulin-dependent diabetes mellitus (3). In addition, tumor-associated antigen-specific TH1 cells can inhibit tumor growth (2, 4–6). However, the exact mode of action, as well as the sites of in vivo homing, proliferation, and trafficking kinetics remain unknown for most immune cells; therefore, noninvasive imaging tools are required to visualize their location and trafficking patterns to enable organ-specific temporal quantification.For in vivo investigation of physiological lymphocyte trafficking, it is strictly required to establish a labeling method with little or no impairment to lymphocyte viability and functionality. One frequently used cell-labeling compound for PET is [⁶⁴Cu]Pyruvaldehyde bis(N⁴-methylthiosemicarbazone) ([⁶⁴Cu]PTSM), which has been applied to mouse lymphocytes, primate stem cells, and human dendritic cells (7–9). We recently established a [⁶⁴Cu]PTSM-labeling protocol that minimizes the harmful effects on T-cell functions (10). Nevertheless, intracellular [⁶⁴Cu]PTSM labeling continues to impair viability and functionality as well as to induce apoptosis/necrosis and DNA damage in TH1 cells. The use of T-cell receptor (TCR)-specific radiolabeled monoclonal antibodies (mAbs) as a label might minimize the harmful effects of ⁶⁴Cu on lymphocytes compared with [⁶⁴Cu]PTSM labeling.Our study aimed to develop a new, highly specific intracellular labeling strategy for T cells that enables whole-body, noninvasive in vivo T-cell tracking. We targeted the antigen-specific TCR using radiolabeled mAbs. We labeled chicken-ovalbumin-TCR-transgenic TH1 cells (cOVA-TCRtg-TH1) with ⁶⁴Cu-DOTA–modified cOVA-TCR–specific mAbs in vitro and investigated the endocytosis-dependent intracellular accumulation of the mAb–TCR complex. We performed temporal and quantitative PET imaging of systemically transferred and labeled cOVA-TCRtg-TH1 cells in a mouse model of OVA-induced acute airway delayed-type hypersensitivity reaction (DTHR) using chicken, turkey (t), and pheasant (ph) OVA to detect cOVA-specific T-cell homing. This labeling approach was successfully transferred to different mouse TCRs and to the expression marker Thy1.2 to label T cells as well as T-cell–depleted lymphocytes of the spleen.     

Sulfiredoxin is an AP-1 target gene that is required for transformation and shows elevated expression in human skin malignancies

Previous studies have shown that a dominant negative form of c-Jun (TAM67) suppresses mouse skin carcinogenesis both in vitro and in vivo. The current study identifies Sulfiredoxin (Srx) as a unique target of activator protein-1 (AP-1) activation and TAM67 inhibition. Manipulation of Srx levels by ShRNA or over-expression demonstrates that Srx is critical for redox homeostasis through reducing hyperoxidized peroxiredoxins. In JB6 cells, knockdown of Srx abolishes tumor promoter-induced transformation and enhances cell sensitivity to oxidative stress. Knockdown of Srx also impairs c-Jun phosphorylation, implicating a role for Srx in the feedback regulation of AP-1 activity. Screening of patient tissues by tissue microarray reveals elevated Srx expression in several types of human skin cancers. Our study indicates that Srx is a functionally significant target of AP-1 blockade that may have value in cancer prevention or treatment.     

Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development

Pancreatic cancer is a disease with an extremely poor prognosis. Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a proapoptotic stress-induced p53 target gene. In this article, we show by immunohistochemical analysis that TP53INP1 expression is dramatically reduced in pancreatic ductal adenocarcinoma (PDAC) and this decrease occurs early during pancreatic cancer development. TP53INP1 reexpression in the pancreatic cancer-derived cell line MiaPaCa2 strongly reduced its capacity to form s.c., i.p., and intrapancreatic tumors in nude mice. This anti-tumoral capacity is, at least in part, due to the induction of caspase 3-mediated apoptosis. In addition, TP53INP1(-/-) mouse embryonic fibroblasts (MEFs) transformed with a retrovirus expressing E1A/ras(V12) oncoproteins developed bigger tumors than TP53INP1(+/+) transformed MEFs or TP53INP1(-/-) transformed MEFs with restored TP53INP1 expression. Finally, TP53INP1 expression is repressed by the oncogenic micro RNA miR-155, which is overexpressed in PDAC cells. TP53INP1 is a previously unknown miR-155 target presenting anti-tumoral activity.     

Structural pedicled mucochondral‐osteal nasoseptal flap: a novel method for orbital floor reconstruction after sinonasal and skull base tumor resection

Unrepaired orbital floor defects after sinonasal and skull‐base tumor resection can lead to herniation of orbital contents into the maxillary or ethmoidal sinuses, possibly resulting in infection and significant cosmetic and functional deficits. Orbital floor defects are usually repaired using prosthetic implants or autogenous material. Nasal septal cartilage has been used previously as a free graft for reconstruction. However, its reliance on local vascular supply can result in ischemia and necrosis in the postoperative period. The vascularized pedicled nasoseptal flap, consisting of mucoperichondrium and mucoperiosteum, is routinely used as an effective reconstruction method for endoscopic repair of cerebrospinal fluid leaks arising from skull base dural defects. However, this flap does not provide rigid structural reconstruction when used alone. We report a case of an orbital floor defect repaired using a pedicled mucochondral‐osteal nasoseptal flap. This technique incorporates the structural component of the nasal septal cartilage and bone with the vascularized pedicled nasoseptal flap. This repair technique may be useful in patients requiring postoperative radiotherapy.     

Induction of Fas (CD95/APO-1) ligand is essential for p53-dependent apoptosis in an in vitro renal carcinoma model system

Purpose The Fas/CD95/APO-1 ligand (FasL) is a death cytokine that binds to cell surface Fas/CD95/APO-1 receptor, yet a possible role of FasL expression in p53-dependent apoptosis is not fully understood in many human malignancies, including renal carcinoma. Methods By Northern blot and Western blot analyses, we determined the effect of p53 on the FasL and Fas receptor expression. To do this, we employed an in vitro renal carcinoma model system that was previously established by stably co-transfecting a temperature-sensitive mutant allele of the p53 tumor suppressor (ts-p53) with either the c-Myc oncogene or adenovirus E1A oncogene in baby rat kidney (BRK) epithelial cells. The ts-p53 is activated only at a permissive temperature. The transactivation activity of p53 was assessed by luciferase reporter assays. The sub-G1 cell population in the cell cycle representing apoptotic cell death was measured by flow cytometric analysis. Results We found that the level of endogenous FasL, but not Fas receptor, was increased at a permissive temperature with delayed kinetics when compared with p21WAF1 expression, but was coincident with p53-induced apoptosis, whereas an apoptosis-defective mutant p53, which lacks the PxxP region (P: Proline, x: any amino acid), failed to induce FasL expression and hence apoptosis. Notably, p53-induced apoptosis was completely blocked by overexpressing a dominant negative inhibitor of the FADD/Mort-1, a pro-apoptotic adaptor that lies immediately downstream of the FasL/Fas receptor. Conclusions These results suggest that the FasL is a critical downstream effector of p53-dependent apoptosis in a cultured BRK renal carcinoma model system.     

Mitochondrial metabolism is essential for invariant natural killer T cell development and function

Conventional T cell fate and function are determined by coordination between cellular signaling and mitochondrial metabolism. Invariant natural killer T (iNKT) cells are an important subset of “innate-like” T cells that exist in a preactivated effector state, and their dependence on mitochondrial metabolism has not been previously defined genetically or in vivo. Here, we show that mature iNKT cells have reduced mitochondrial respiratory reserve and iNKT cell development was highly sensitive to perturbation of mitochondrial function. Mice with T cell-specific ablation of Rieske iron-sulfur protein (RISP; T-Uqcrfs1^−/−), an essential subunit of mitochondrial complex III, had a dramatic reduction of iNKT cells in the thymus and periphery, but no significant perturbation on the development of conventional T cells. The impaired development observed in T-Uqcrfs1^−/− mice stems from a cell-autonomous defect in iNKT cells, resulting in a differentiation block at the early stages of iNKT cell development. Residual iNKT cells in T-Uqcrfs1^−/− mice displayed increased apoptosis but retained the ability to proliferate in vivo, suggesting that their bioenergetic and biosynthetic demands were not compromised. However, they exhibited reduced expression of activation markers, decreased T cell receptor (TCR) signaling and impaired responses to TCR and interleukin-15 stimulation. Furthermore, knocking down RISP in mature iNKT cells diminished their cytokine production, correlating with reduced NFATc2 activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in iNKT cell development and activation outside of its traditional role in supporting cellular bioenergetic demands.

Cellular metabolic pathways are interwoven with traditional signaling pathways to regulate the function and differentiation of T cells (1–3). Upon activation, effector T cells display a marked increase in glycolytic metabolism even in the presence of ample oxygen, termed aerobic glycolysis (4). We have previously shown that despite increased aerobic glycolysis, T cell activation depends on mitochondrial metabolism for generation of reactive oxygen species (ROS) for signaling (5). As activated T cells progress to a memory or regulatory phenotype, they preferentially oxidize fatty acids to support mitochondrial metabolism, and enhanced fatty acid oxidation (FAO) and spare respiratory capacity (SRC) are essential to maintenance of their phenotype (6, 7).CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of lymphocytes that exhibit a preactivated phenotype with rapid effector responses (8, 9). iNKT cells are capable of producing large amount of proinflammatory and antiinflammatory cytokines thus have broad immunomodulatory roles (8–10). Given that these cells are poised for rapid proliferation and cytokine production, we hypothesized that coordination of cellular signaling with cellular metabolism will be especially critical for optimal iNKT function. In support of this hypothesis, several studies suggest that modulation of cellular metabolism affects iNKT cell development and function. iNKT cell development is diminished upon deletion of the miR-181 a1b1 cluster, which regulates phosphoinositide 3-kinase signaling and decreases aerobic glycolysis (11, 12). In addition, T cell-specific deletion of Raptor (a component of mTORC1), a metabolic regulator, leads to defects in iNKT cell development and function (13, 14). Loss of folliculin-interacting protein 1 (Fnip1), an adaptor protein that physically interacts with AMP-activated protein kinase, also results in defective NKT cell development, and interestingly conventional T cells develop normally (15). Furthermore, a number of studies targeting bioenergetics processes or related molecules, like alteration of glucose metabolism, mitochondrial-targeted antioxidant treatment, and receptor-interacting protein kinase 3-dependent activation of mitochondrial phosphatase, showed significant effects on iNKT cell ratio and function (16–19). A recent study showed that iNKT cells are less efficient in glucose uptake than CD4⁺ T cells. Furthermore, activated iNKT cells preferentially metabolize glucose by the pentose phosphate pathway and mitochondria, instead of converting into lactate, since high lactate environment is detrimental to their homeostasis and effector function (20).In conventional lymphocytes, mitochondria clearly play a role in coordination of cell signaling and cell fate decisions outside of production of energy (5, 21–23). During T cell activation mitochondria localize at immune synapses that T cells form with antigen-presenting cells (22). T cell receptor (TCR) stimulation triggers mitochondrial ROS (mROS) production as well as mitochondrial ATP production that are released at the immune synapses and are critical for Ca²⁺ homeostasis and modulation of TCR-induced downstream signaling pathways (22). We previously showed that mice with T-cell–specific deletion of Rieske iron sulfur protein (RISP), a component of mitochondrial complex III of the mitochondrial electron transport chain (ETC), are defective in antigen-specific T cell activation due to deficiency of mROS required for cellular signaling (5). Several recent studies showed that ROS or factors that affect ROS production are also important in iNKT cell development and effector functions (24–27). In addition, inhibition of mitochondrial oxidative phosphorylation (OXPHOS) by oligomycin has been shown to decreased survival and cytokine production by splenic iNKT cells (20). However, the requirement of mitochondrial metabolism for iNKT cell development and function has not been previously defined genetically or in vivo.Here we showed that iNKT cells have comparable basal mitochondrial oxygen consumption to conventional T cells but displayed lower SRC and FAO, which are thought to impart cells with mitochondrial reserve under stress. Using Uqcrfs1^fl/fl;CD4-Cre⁺ (hereafter referred as T-Uqcrfs1^−/−) mice, we showed that abrogation of mitochondrial metabolism resulted in a cell-autonomous defect in iNKT cell development in thymus and periphery. The iNKT cells were able to proliferate but exhibited impaired activation, suggesting that they were not lacking bioenergetically but rather had aberrant TCR signaling in vivo, leading to altered expression of downstream factors required for their terminal maturation. Accordingly, T-Uqcrfs1^−/− iNKT cells displayed lower T-bet and CD122 levels and did not respond to interleukin (IL)-15 stimulation. Knockdown of RISP in mature iNKT cells also limited NFATc2 translocation to the nucleus. Collectively, our data highlighted an important role of mitochondrial metabolism in modulating TCR signaling in vivo and regulating iNKT cell development and function.     

The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2–1 mutant. The pas2–1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes.     

In vitro and in vivo investigations for the development of cytostatic methylhydrazones

Summary In in vitro short-term (3 h) assays, the -chloroethyl-methyl-hydrazones B 1 and B 2 inhibit the uptake of ³H-thymidine by EAC and L 1210 leukemia cells, B 2 being 5 to 10 times more effective than B 1. The growth inhibitory effect of both compounds was also confirmed in long-term (7 days) clonal assays using agar-containing glass capillaries, B 2 again being more effective than B 1. In contrast to these differences in vitro, in vivo both substances showed remission to the same degree in EAC- and complete resistance in L 1210-bearing mice. The diverging in vitro/in vivo sensitivities were thought to result from differences in the affinity of the methylhydrazones to the tumor cells: using short exposure periods (3 h) B 1 was more inhibitory than B 2 on both EAC and L 1210 colony growth; i.e., the more hydrophilic B 2 could more easily be washed off. To further test the idea of different cell membrane affinities, the methylhydrazones ZB 1 and P 1 with increasing lipophilic properties were synthesized. In vitro, after both pulse and continuous exposure ZB 1 and P 1 showed enforced inhibitory effects on colony growth. In vivo, ZB 1 and P 1 reduced the tumor weight of EAC mice, while only P 1 increased the survival time of L 1210 mice. The results suggest that from the combination of in vitro/in vivo assays mechanistic conclusions can be derived that are valuable for further development of these cystostatics.Abbreviations B 1 N-methyl-N--chloroethyl-benzaldehyde-hydrazone - B 2 N-methyl-N--chloroethyl(p-dimethylamino)benzaldehyde-hydrazone - EAC Ehrlich Ascites Carcinoma - FCS fetal calf serum - ³H-dThd 6-³-H-thymidine - L 1210 Leukemia L 1210 - P 1 N-methyl-N--chloroethyl-pentadiene-benzaldehyde-hydrazone - ZB 1 N-methyl-N--chloroethyl-cinnamoylaldehyde-hydrazone     

STAT1 is overexpressed in tumors selected for radioresistance and confers protection from radiation in transduced sensitive cells

Nu61, a radiation-resistant human tumor xenograft, was selected from a parental radiosensitive tumor SCC-61 by eight serial cycles of passage in athymic nude mice and in vivo irradiation. Replicate DNA array experiments identified 52 genes differentially expressed in nu61 tumors compared with SCC-61 tumors. Of these, 19 genes were in the IFN-signaling pathway and moreover, 25 of the 52 genes were inducible by IFN in the nu61 cell line. Among the genes involved in IFN signaling, STAT1alpha and STAT1beta were the most highly overexpressed in nu61 compared to SCC-61. STAT1alpha and STAT1beta cDNAs were cloned and stably transfected into SCC-61 tumor cells. Clones of SCC-61 tumor cells transfected with vectors expressing STAT1alpha and STAT1beta demonstrated radioprotection after exposure to 3 Gy (P < 0.038). The results indicate that radioresistance acquired during radiotherapy treatment may account for some treatment failures and demonstrate an association of acquired tumor radioresistance with up-regulation of components of the IFN-related signaling pathway.     

Crosses between diabetic BB/OK and wild rats confirm that a third gene is essential for diabetes development

Several crossing studies with diabetic BB rats have shown that in addition to the lymphopenia (Iddm1) and the MHC class II genes of the RT1^u haplotype (Iddm2) there are further non-MHC genes essential for diabetes development. Because diabetes-resistant inbred rat strains may be homozygous for one of the diabetogenic non-MHC genes, masking the expression of diabetogenic genes and leading to an underestimation of the number of diabetogenic genes, we crossed wild and diabetic BB/OK rats. The F1 hybrids were backcrossed onto diabetic female (BC1W-F, n=97) and male BB/OK rats (BC1W-M, n=98) transferred to a specified-pathogen-free environment and studied for the frequency and age at onset of diabetes up to an age of 30 weeks. Comparing the results of these BC1 W hybrids with similarly derived hybrids using diabetes-resistant DA rats (BC1DA-F, n=113; BC1DA-M, n=216), the diabetes frequency in total was comparable indicating the action of three recessive genes. The percentage of diabetics in Iddm1 and Iddm2 homozygotes confirmed the existence of the third gene, Iddm3, but there were some sex differences; significantly more male than female BC1W-F and significantly more BC1DA-M than BC1DA-F males were diabetic. Regarding the age at onset, the BC1W-F hybrids manifested not only significantly earlier, but also more uniformly than BC1DA-F and BC1-M hybrids. Received: 24 September 1997 / Accepted in revised form: 11 March 1998