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Introduction: Integrating additional factors into the International Federation of Gynecology and Obstetrics (FIGO) staging system is needed for accurate patient classification and survival prediction. In this study, we tested machine learning as a novel tool for incorporating additional prognostic parameters into the conventional FIGO staging system for stratifying patients with epithelial ovarian carcinomas and evaluating their survival., Material and Methods: Cancer-specific survival data for epithelial ovarian carcinomas were extracted from the Surveillance, Epidemiology, and End Results (SEER) program. Two datasets were constructed based upon the year of diagnosis. Dataset 1 (39 514 cases) was limited to primary tumor (T), regional lymph nodes (N) and distant metastasis (M). Dataset 2 (25 291 cases) included additional parameters of age at diagnosis (A) and histologic type and grade (H). The Ensemble Algorithm for Clustering Cancer Data (EACCD) was applied to generate prognostic groups with depiction in dendrograms. C-indices provided dendrogram cutoffs and comparisons of prediction accuracy., Results: Dataset 1 was stratified into nine epithelial ovarian carcinoma prognostic groups, contrasting with 10 groups from FIGO methodology. The EACCD grouping had a slightly higher accuracy in survival prediction than FIGO staging (C-index = 0.7391 vs 0.7371, increase in C-index = 0.0020, 95% confidence interval [CI] 0.0012-0.0027, p = 1.8 × 10 -7 ). Nevertheless, there remained a strong inter-system association between EACCD and FIGO (rank correlation = 0.9480, p = 6.1 × 10 -15 ). Analysis of Dataset 2 demonstrated that A and H could be smoothly integrated with the T, N and M criteria. Survival data were stratified into nine prognostic groups with an even higher prediction accuracy (C-index = 0.7605) than when using only T, N and M., Conclusions: EACCD was successfully applied to integrate A and H with T, N and M for stratification and survival prediction of epithelial ovarian carcinoma patients. Additional factors could be advantageously incorporated to test the prognostic impact of emerging diagnostic or therapeutic advances., (© 2021 The Authors. Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).)

Purpose: Ovarian epithelial carcinomas, including the predominant serous ovarian carcinoma (SOC) type, are heterogeneous malignancies. Even though invasive SOCs of low and high grade can be distinguished by morphology and molecular or immunohistochemical profiles, age-specific risks relevant to their separate carcinogenic pathways and clinical features have not been fully explored., Methods: In search of further clues to the etiology/pathogenesis of low-grade and high-grade SOCs, we analyzed incidence rate patterns. Case and age-adjusted population data were obtained from the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) program for years 1990 through 2005. Descriptive epidemiology for n = 19,899 cases was supplemented with age-period-cohort models fitted by grade., Results: SOC age-adjusted incidence rate ratios (IRR) of high to low grade (IRR(H/L)) were <1.0 before age 40, and >1.0 thereafter. Accordingly, SOC age-specific incidence rates were also greater for low grade before age 40 years, and then greater for high grade. The reversals of IRR(H/L), with crossings of the age-specific incidence rate near age 40 years occurred irrespective of early or late SOC stage. These results were reproducible and reliable in age-period-cohort models that were adjusted for period and cohort effects (P approximately 0 for age interactions by grade)., Conclusions: Robust qualitative age interactions between low-grade and high-grade SOC showed that grade is an age-specific effect modifier in these malignancies. With increasing research interest in identifying the genomic determinants of SOC risk, therapeutic response, and outcome, future analytic studies and clinical trials should be powered to account for age-dependent grade interactions.

The protein kinase C (PKC) family of enzymes is a regulator of transmembrane signal transduction, and involvement of some PKC isoforms in T-cell activation has been demonstrated. Nevertheless, very little is known about their involvement in the Amyloid beta (Abeta)-dependent molecular signals in the T lymphocytes of Alzheimer disease (AD) patients. Therefore, the aim of this study was to investigate the involvement of PKC-alpha, PKC-delta and PKC-zeta expression and activity in the signaling machinery activated in Abeta-reactive T cells, in adult healthy individuals, elderly healthy subjects, and from patients with AD. The results show that in peripheral T-cells from early AD patients, Abeta(1-42) produced a distinct subpopulation highly expressing P-PKC-delta, while in severe AD patients the same treatment induced two distinct P-PKC-delta and P-PKC-zeta T-cell subpopulations. Such subpopulations were not noticeable following CD3/CD28 treatment of the same samples or after treatment of peripheral T cells from healthy adult or elderly subjects with Abeta(1-42) or with CD3/CD28. We believe that these findings may be of help in possible attempts to develop further diagnostic strategies useful for the characterization of AD.

Peroxiredoxins (Prx) comprise an extended family of small antioxidant proteins which conserve a thioredoxin-dependent catalytic function that can contribute to cell protection from reactive oxygen species (ROS). ROS generation is one of the deleterious intracellular effects of ionizing radiation, but the role of Prx during radiation treatment has not been extensively explored. Present experiments measure effects of ionizing radiation on expression of human Prx types I (PAGA), II (NKEF-B) and IV (AOE372) in human myeloid leukemia cells (K562). Prx gene transcription was analyzed by amplifying with RT-PCR cDNAs complementary to each Prx-specific coding sequence and by identifying the derived products with Southern blotting procedure. Transcripts of GAPDH were used as the endogenous standard for semi-quantitative comparisons. No consistent increase in Prx gene expression was detected at time intervals up to 72 h after gamma radiation doses that caused cell cycle arrest and nuclear damage (maximum 20 Gy). Immunoblots also were consistent with a prolonged expression or stability of the Prx I/II proteins. Similarly, a cytotoxic concentration of the oxidant hemin, which stimulates rapid hemoglobinization of K562 cells, caused no induction of Prx gene expression. Our results indicate a high Prx stability in human radio-resistant leukemia cells.

The relationship of plasma membrane biophysical properties to the anti-proliferative effect of interferon-alpha (IFN-alpha) was investigated in Daudi lymphoblasts cell lines with sensitivity to growth inhibition, parallel clonal variants selected for resistance, and one revertant subclone. Lateral mobility of surface differentiation antigens (I2, CD19, CD20, and sIgM-kappa) were measured by fluorescence recovery after photobleaching (FRAP). The mean diffusion coefficients, D, values for two clones of IFN-alpha resistant Daudi cells were significantly higher (D = 8.1-11 x 10(-10) cm2/sec) than for parental sensitive cells (D = 4.9-7.4 x 10(-10) cm2/sec). Microviscosity of the plasma membranes were probed by electron spin resonance (ESR) spectrometry. These results also indicate a greater degree of molecular motional freedom in resistant cells. Treatment of sensitive lymphoblasts with IFN-alpha (100-400 U/10(6) cells) for 5-30 min consistently increased mean values of D and the degree of spin-probe motional freedom, whereas no significant differences were detected in resistant cells. The effect of IFN-alpha on the membrane potential (Em) of Daudi cells was quantitated by flow cytometry using a voltage-sensitive oxonol dye. Membrane potential of all clones was similar (-50 to -56 mV). Treatment with IFN-alpha for 8-10 min caused hyperpolarization in the sensitive cells (deltaEm up to 45 mV), but only minimal hyperpolarization in the resistant ones (deltaEm up to 7 mV). We concluded that sensitivity to IFN-alpha and treatment with IFN-alpha are related to the biophysical status of plasma membranes.

Previous results demonstrated that the occurrence of death in human peripheral B lymphocytes by TNF-alpha was paralleled by the activation of the cytoplasmic Jak1 and Tyk2 protein kinases, along with the recruitment of transcription factors Stat3 and Stat5b. In this study we demonstrate that the balance of survival signals in the presence of TNF-alpha was altered by the addition of a salicylate compound, the endonuclease inhibitor aurintricarboxylic acid (ATA). Apoptosis effected by TNF-alpha alone was suppressed by ATA and this event was paralleled by phosphorylation and nuclear translocation of Jak2, Stat2, Stat4 and NF-kB, along with inhibition of caspase activation. These results confirm that among the different cellular responses evoked by TNF-alpha in human B cells, recruitment of Jak/Stat proteins and possible related gene modulation represent contributing factors and address the issue of the development of potential therapeutic strategies aimed at the control of systemic or local effects produced by TNF-alpha.

Understanding how normal and immortalized bronchial epithelial cells respond to modulators of gap junctional communication will increase our understanding of the process of tumor promotion. In the present study we compared to effects of retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the rate of fluorescent dye transfer via gap junctions in primary human tracheo-bronchial epithelial cells (TBE) and SV40 large T-antigen immortalized, non-tumorigenic bronchial epithelial cells (BEAS-2B). RA in the physiological range (0.001-1 microM) inhibited cell proliferation (DNA synthesis, mitotic index) more in primary TBE cells than BEAS-2B cells. Also in RA-treated cells, decreased cell proliferation was coupled to decreased gap junctional communication (GJC) in TBE but not in BEAS-2B cells. TPA strongly suppressed GJC and proliferation in primary TBE cells, whereas BEAS-2B exhibited increased GJC and retained a significant fraction of cells undergoing DNA synthesis. Our studies show that an uncoupling of GJC and cell proliferation is associated with a differential response to the growth inhibitory effects of RA and phorbol esters in immortalized compared to primary human bronchial epithelial cells., (Copyright 2002 Elsevier Science.)

This article provides an overview of arbovirus diseases from the perspective of laboratory diagnosis and related responsibilities of health personnel. Although the disease manifestations are very diverse, general lessons relevant to optimal use of laboratory resources can be drawn from medically important examples. This approach is warranted, because under conditions of bioterrorism or emergence of a novel pathogen, ecologic clues that ordinarily facilitate identification of an arbovirus infection most likely will be absent.

The transcription factor Stat5a critically mediates prolactin (PRL)-induced mammary gland development and lactogenesis. PRL also stimulates growth and differentiation of prostate tissue. Specifically, hyperprolactinemia gives rise to prostate hyperplasia, and prostate size is reduced in PRL-deficient mice. We therefore investigated the importance of Stat5a for prostate development and function by examining Stat5a-null mice. The absence of Stat5a in mice was associated with a distinct prostate morphology characterized by an increased prevalence of local disorganization within acinar epithelium of ventral prostates. Affected acini were typically filled with desquamated, granular epithelial cells that had become embedded in dense, coagulated secretory material. These features were reminiscent of acinar cyst formation and degeneration frequently observed in human benign prostate hyperplasia, however, cystic changes in prostate acini of Stat5a-deficient mice were not associated with increased prostate size or morphologic hallmarks of epithelial hyperplasia. Instead, immunohistochemistry of the prostate-specific secretory marker, probasin, suggested that hypersecretory function of the epithelium could underlie local congestion and cyst formation in prostates of Stat5a-null mice. Serum testosterone and PRL levels were normal in Stat5a knockout mice, but prostate PRL receptor expression was reduced as determined by immunohistochemistry. Expression levels or activation states of other PRL signal transduction proteins, including Stat5b, Stat3, Stat1, ERK1, and ERK2 were not altered. The present study offers the first evidence for a direct role of Stat5a in the maintenance of normal tissue architecture and function of the mouse prostate.

Stat5a and Stat5b are discretely encoded transcription factors that mediate signals for a broad spectrum of cytokines. Their activation is often an integral component of redundant cytokine signal cascades involving complex cross-talk and pleiotropic gene regulation by Stat5 has been implicated in cellular functions of proliferation, differentiation and apoptosis with relevance to processes of hematopoiesis and immunoregulation, reproduction, and lipid metabolism. Although Stat5a and Stat5b show peptide sequence similarities of > 90%, targeted gene disruptions in mice yield distinctive phenotypes. Prolactin-directed mammary gland maturation fails without functional Stat5a, while disruption of Stat5b in males mitigates growth hormone effects on hepatic function and body mass. The molecular basis for this biologic dichotomy is probably multifaceted. Limited structural dissimilarities between the Stat5a and Stat5b transactivation domains, or subtle differences in the DNA-binding affinities of Stat5 dimer pairs undoubtedly influence gene regulation, but cell-dependent asymmetries in availability of phosphorylated Stat5 can be an underlying factor. Differences in serine phosphorylation(s) of Stat5a and Stat5b, or Stat5 associations with adaptor proteins or co-transcription factors are other potential sources of functional disparity and the signal amplitude, frequency or duration also can be significant. In addition to Stat5 signal attenuation by phosphatase actions or classical feedback inhibition, truncated forms of Stat5 lacking in transactivation capacity may compete upstream for activation and diminish access of full length molecules to DNA binding sites.

In a manner similar to many other cytokines, treatment of cells with granulocyte CSF (G-CSF) has been shown to induce the tyrosine phosphorylation of the STAT proteins. Activation of Stat1 and Stat5 by G-CSF requires the membrane-proximal cytoplasmic domain of the receptor, including box1 and box2, while G-CSF-stimulated tyrosine phosphorylation of Stat3 also requires a region distal to box 2. In this study, we show that although the membrane-proximal 55 amino acids of the G-CSF receptor are sufficient for activation of Stat5, the maximal rate of Stat5 activation requires an additional 30 amino acids of the cytoplasmic domain. In contrast, the distal carboxyl-terminal region of the receptor appears to down-regulate Stat5 activation in that deletion of this carboxyl terminus results in increased amplitude and prolonged duration of Stat5 activation by G-CSF. Significantly, expression of a truncated dominant-negative Stat5 protein in hemopoietic cells not only inhibits G-CSF-dependent cell proliferation, but also suppresses cell survival upon G-CSF withdrawal. We further show that a potential protein tyrosine phosphatase may play a critical role in the down-regulation of G-CSF-stimulated Stat5 activation. These results demonstrate that two distinct cytoplasmic regions of the G-CSF receptor are involved in the regulation of the intensity and duration of Stat5 activation, and that Stat5 may be an important player in G-CSF-mediated cell proliferation and survival.

Type I interferons (IFN alpha and IFN beta) are presently used in the adjuvant treatment of several human cancers. However, these cytokines have demonstrated only modest success in breast cancer therapy, and research efforts have focused on improving their efficacy. Recent progress in understanding the molecular mechanisms underlying the antiproliferative effects of IFNs has identified the cytoplasmic transcription factor Stat1 as a critical mediator. It is, therefore, possible that IFN-induced growth inhibition of mammary epithelial cells is counteracted by other cytokines that also use Stat1. One such candidate IFN-antagonist with particular relevance to breast cancer is the mammotropic hormone prolactin (PRL). The main goal of this study was to examine whether PRL would interfere with type I IFN (IFN alpha/beta) signal transduction by competing for limited cytoplasmic Stat factors. A second aim was to test whether pretreatment of mammary tumor cell lines with IFN gamma could enhance the effect of IFN alpha/beta. By analyzing the effect of PRL on IFN alpha/beta-induced tyrosine phosphorylation of Stat proteins and their binding to IFN-regulated genes, we now report that costimulation of PRL receptors did not interfere with IFN alpha/beta signals in several human breast cancer cell lines, including T47D, MCF-7, and BT-20. Specifically, PRL did not affect IFN alpha/beta-induced tyrosine phosphorylation or heterodimerization of Stat1 and Stat2 in any cell line. Instead, IFN alpha/beta- and PRL-induced tyrosine phosphorylation of Stat1 was additive and occurred without evidence of competition for limited concentrations of cytoplasmic Stat1. A similar additive relationship was observed on IFN alpha/beta- and PRL-induced Stat3 tyrosine phosphorylation. Furthermore, electrophoretic mobility shift assays showed that type I IFNs induced predominantly Stat1-Stat2 or Stat1-Stat3 heteromeric complexes with various IFN-response elements of IFN-stimulated genes, whereas PRL induced Stat1 homodimers. Despite significant mutual use of Stats by IFNs and PRL, these results indicated a high degree of signaling specificity in the two receptor systems, and that cytoplasmic levels of Stat proteins were not limiting. Similarly, PRL did not interfere with the growth-inhibitory effect of IFN beta. On the other hand, the study indicated that pretreatment of human breast cancer cell lines with IFN gamma enhanced the growth-inhibitory action of type I IFNs, suggesting a possible avenue for improving the effect of type I IFNs in the treatment of breast cancer patients.

Multiple biologic effects of interferon-alpha (IFN-alpha), including cell growth inhibition and antiviral protection, are initiated by tyrosine phosphorylation of STAT proteins. Although this signal pathway has been intensively investigated, the relevance of STAT signal persistence has received scant attention. Using paired isogenic lymphoma cells (Daudi), which either are sensitive or resistant to growth inhibition by IFN-alpha, we found comparable initial tyrosine phosphorylation of multiple STAT proteins; however, the phosphorylation durations and associated DNA-binding activities diverged. Phosphorylation and DNA-binding capacity of STAT1 decreased after 4 to 8 hours in resistant cells, as compared with 24 to 32 hours in sensitive cells, whereas phosphorylation of STAT3 and STAT5b was briefer in both lines. Functional significance of the prolonged STAT1 signal, therefore, was explored by experimental interruption of tyrosine phosphorylation, either by premature withdrawal of the IFN-alpha or deferred addition of pharmacologically diverse antagonists: staurosporine (protein kinase inhibitor), phorbol 12-myristate 13-acetate (growth promoter), or aurintricarboxylic acid (ligand competitor). Results indicated that an approximately 18-hour period of continued STAT1 phosphorylation was associated with growth arrest, but that antiviral protection developed earlier. These differences provide novel evidence of a temporal dimension to IFN-alpha signal specificity and show that duration of STAT1 activation may be a critical variable in malignant cell responsiveness to antiproliferative therapy.

Biological effects of many hormones and cytokines are mediated through receptor-associated Jak tyrosine kinases and cytoplasmic Stat transcription factors, including critical physiological processes such as immunity, reproduction, and cell growth and differentiation. Pharmaceuticals that control Jak-Stat pathways are therefore of considerable interest. Here we demonstrate that a single Jak-Stat pathway can be activated by aurintricarboxylic acid (ATA), a negatively charged triphenylmethane derivative (475 Da) with anti-apoptotic properties. In prolactin (PRL)-dependent Nb2 lymphocytes, ATA sustained cell growth in the absence of hormone and mimicked rapid PRL-induced tyrosine phosphorylation of Jak2 and activation of Stat5a and Stat5b with tyrosine phosphorylation, heterodimerization, DNA binding, and induction of the Stat5-regulated pim-1 protooncogene. ATA also mimicked PRL activation of serine kinases ERK1 and ERK2. However, unlike PRL, ATA did not regulate Stat1 or Stat3. ATA also did not affect Jak3, which is activated in these cells by interleukin-2 family cytokines. Although the mechanism and specificity by which ATA activates Jak2, Stat5, and ERKs in Nb2 cells are still unclear, the present study demonstrates that certain hormone or cytokine effects on Jak-Stat pathways can be discretely imitated by a low molecular weight, non-peptide pharmaceutical. The results are also consistent with Stat5 involvement in lymphocyte growth and survival.

We found that simian virus 40 (SV40) induces mesotheliomas in hamsters and that 60% of human mesotheliomas contain and express SV40 sequences, results now confirmed by others [ref. 3-5, and presentations by D. Griffiths & R. Weiss, F. Galateau-SallE, and H.I.P. at "Simian virus 40: A possible human polyoma virus," NIH workshop, 27-28 January 1997, Bethesda, MD (transcript available through SAG Corp., Washington, DC 20008)]. Mesothelioma, an aggressive malignancy resistant to therapy, originates from the serosal lining of the pleural, pericardial and peritoneal cavities. The incidence of mesothelioma continues to increase worldwide because of exposure to crocidolite asbestos. However, at least 20% of mesotheliomas in the United States are not associated with asbestos exposure, and only a minority of people exposed to high concentrations of asbestos develop mesothelioma. Thus, other carcinogens may induce mesothelioma in individuals not exposed to asbestos, and/or may render particular individuals more susceptible to the carcinogenic effect of asbestos. We investigated whether the expression of the SV40 large T-antigen (Tag) interferes with the normal expression of the tumor suppressor gene p53 in human mesotheliomas. We found that SV40 Tag retains its ability to bind and to inactivate p53, a cellular protein that when normally expressed plays an important role in suppressing tumor growth and in inducing sensitivity to therapy. Our findings do not establish a cause-and-effect relation, but indicate that the possibility that SV40 contributes to the development of human mesotheliomas should be carefully investigated.

Transcription factors of the Stat gene family are selectively activated by many hormones and cytokines. Stat5 originally was cloned as a prolactin-stimulated DNA-binding protein, but is also activated by non-lactogenic cytokines in many cell types. The recent identification of two distinct Stat5 genes, which encode a 94-kDa Stat5a and a 92-kDa Stat5b as well as several lower molecular weight isoforms, suggests additional complexity and combinatorial possibilities for transcriptional regulation. We now report a biochemical analysis of prolactin activation of Stat proteins in Nb2 lymphocytes, which was associated with: 1) rapid tyrosine phosphorylation of Stat5a, Stat5b, a COOH-terminally truncated 80-kDa Stat5 form, Stat1alpha, and Stat3; 2) rapid and selective formation of Stat5a/b heterodimers, without involvement of Stat1alpha or Stat3; 3) marked serine, but not threonine phosphorylation of Stat5a and Stat5b; and 4) the appearance of two qualitatively distinct Stat5 protein complexes, which discriminated between oligonucleotides corresponding to the prolactin response elements of the beta-casein and interferon regulatory factor-1 gene promoters. Collectively, our analyses showed that Stat5a and Stat5b respond similarly to prolactin receptor activation, but also suggested that the two genes have evolved unique properties that may contribute to the specificity of receptors that utilize Stat5 signaling proteins.

Alpha and gamma interferons rapidly induce several early response genes in primary human diploid fibroblasts. The transcription rates of these genes are maximal after 1 h of interferon treatment and return to basal levels within 8 h. Three different interferon-activated DNA-binding complexes (ISGF3, GAF, and FcRF gamma) that are responsible for transcriptional activation of cellular genes have been characterized. Assembly of these complexes requires tyrosine phosphorylation of one or more of the protein components. In this report, we demonstrate that a nuclear tyrosine phosphatase is responsible for the deactivation of these interferon-regulated transcription factors and the subsequent transcriptional downregulation of the corresponding genes. Furthermore, tyrosine phosphorylation is required for nuclear localization of the 91-kDa protein that is part of all three interferon-induced transcription complexes. These results provide the first evidence for a nuclear tyrosine phosphatase activity as a mechanism of transcriptional regulation.

Several clinical protocols are attempting to utilize the combined anti-proliferative signal of interferon-alpha (INF-alpha) and tamoxifen on cancer cells. We demonstrated here that the effect of these two agents on the growth of the premacrophage U937 cells is antagonistic. This antagonistic effect is paralleled by the ability of tamoxifen to modulate the INF-alpha-induced hyperpolarization in these cells. INF-alpha-induced hyperpolarization was shown before to be an integral part of the anti-proliferative signal of this agent. Tamoxifen liberates Ca2+ from intracellular stores of U937 cells but this effect of the drug is not the cause of its antagonistic effect with the anti-proliferative signal of IFN-alpha. We suggest therefore, that the combined effect of these two anti-cancer drugs could also be advantageous for macrophage proliferation.

Binding of interferons (IFNs) to their cell surface receptors stimulates rapid translocation of cytoplasmic proteins to the nucleus and the expression of a variety of cellular genes within minutes. Translocated proteins subsequently bind to the interferon-stimulated response element (ISRE) located in the promoters of all IFN-activated cellular genes. We report here that ouabain, a specific inhibitor of the Na/K ATPase, selectively inhibited transcription of several IFN-alpha-induced cellular RNAs under conditions in which some other well-described signal transduction pathways remained intact. The latter included induction of human metallothionein 2A (HMT2A) by phorbol ester and induction of IP-10 RNA by IFN-gamma. Ouabain itself induced RNA of the protooncogene c-fos which conversely was inhibited by IFN-alpha. Specificity of the ouabain effects on IFN alpha-induced RNAs with respect to a direct action on the Na/K ATPase was shown with a transfected monkey CV-1 cell line which expresses the ouabain-insensitive rat alpha 1 subunit. Electrophoretic mobility shift assays (EMSAs) using nuclear extracts from ouabain-treated cells demonstrated that ouabain decreased IFN alpha-induced binding of the ISGF3 complex to the ISRE. Reconstitution experiments showed that this effect of ouabain is not due to the inhibition of IFN alpha activation of the ISGF3 alpha subcomponent, which occurs in the cytoplasm, but a selective depletion of the ISGF3 gamma factor which in concert with activated ISGF3 alpha induces interferon-stimulated gene (54 kDa) transcription. These findings imply that intracellular ion balance can selectively regulate the half-life of the ISGF3 gamma protein or the ability of this protein to complex with ISGF3 alpha to activate IFN alpha-regulated cellular genes.

Cytochalasin B, a drug that alters microfilament structure, was found to modulate interferon-alpha (IFN-alpha)-induced changes in ion fluxes, in motional freedom of spin probes, and lateral diffusion of surface antigens. These changes occur in Daudi cells inherently sensitive to the antiproliferative signal of IFN-alpha, but not in insensitive cells, and were associated with the antiproliferative signal previously. The biophysical effects of cytochalasin B were detected by flow cytometric quantitation of membrane potential using an oxonol dye, by electron spin resonance (ESR) spectrometry, and by measurements of fluorescence recovery after photobleaching (FRAP) of surface antigens using a laser-interactive cell imaging system. Cytochalasin B treatment increased an IFN-alpha-induced membrane potential shift by -5 mV. The motional freedom of 5-doxyl-stearic acid changed from 0.67 to 0.63, as expressed by the order parameter, S, with IFN-alpha treatment and was prevented by cytochalasin B. Changes in the lateral diffusion of surface antigens induced by IFN-alpha treatment, D = 5.3 x 10(-10) without treatment and D = 7.8 x 10(-10) cm2/s with treatment, was blocked by cytochalasin B. In contrast, the microtubule stabilizers taxol and D2O and the microtubule depolymerizing colcemid were ineffective at dose levels sufficient to cause the characteristic cell physiological alterations of these agents. These results implicate microfilaments but not the microtubule system in transduction of the antiproliferative signal by IFN-alpha in Daudi cells.

The effects of transforming growth factor beta (TGF-beta) on cell-to-cell communication were investigated in the log phase of growth in normal BE and in adenovirus-12 SV40 hybrid virus transformed BE cells (strain BEAS-2B). Gap junctions in these cells were identified immunocytochemically. Exposure of BE cells to exogenous TGF-beta (0.04-4.0 pM) in serum-free keratinocyte growth medium (KGM) for 1 or 24 h reduced the rate of fluorescent dye transfer (i.e. cell-to-cell communication) by 30-50% in BE cells. Inversely, in BEAS-2B cells, TGF-beta after 1 h induced a 2- to 10-fold increase in the rate of dye transfer. After 24 h of TGF-beta, communication among BEAS-2B cells was not significantly different from controls (no exogenous TGF-beta). The protein kinase C (PKC) inhibitor H-7 induced a dose-dependent enhancement in communication, which was even higher in the presence of TGF-beta (4 pM X 24 h). The calmodulin antagonist W-7 enhanced communication in BEAS-2B cells independently of the presence of TGF-beta. In keratinocyte basal medium (KBM) supplemented with EGF (5 ng/ml) or with TGF-beta (4.0 pM) dye transfer was reduced or enhanced respectively. The combination of EGF and TGF-beta in KBM antagonized the stimulatory effect of the latter on communication in BEAS-2B cells. In BE cells, continuous exposure (4 days) to TGF-beta in KGM induced a dose-dependent inhibition of proliferation and an increased expression of a keratinized, epidermoid phenotype. This correlated with a reduction in the expression of a mucous secretory phenotype. Increased exposure to TGF-beta (0.04-4.0 pM) decreased the labeling index in BEAS-2B cells, but the cells retained a growth advantage over normal BE cells, and did not express a keratinized epidermoid morphology. With respect to dye transfer as an index of cell-to-cell communication, we conclude (i) that an inhibition or enhancement of communication is involved in the response of bronchial epithelial cells to mitogens (e.g. epidermal growth factor) or growth inhibitors (e.g. TGF-beta), (ii) that PKC and Ca(2+)-calmodulin-dependent processes regulate dye transfer, and (iii) the effects of TGF-beta are mediated by PKC.

In earlier studies, we showed that when interferon alpha (IFN alpha) binds to cell receptors, it alters membrane potentials. In IFN-sensitive Daudi cells derived from Burkitt's lymphoma patients, IFNs produce dose-dependent hyperpolarization, decreased plasma membrane viscosity, modulation of the microfilament system, and altered synthesis of cAMP, cGMP and prostaglandins. No such changes were seen in IFN alpha-resistant Daudi cells. In this study, we found that indomethacin, pentoxifylline, or the cGMP inducer sodium azide (NaN3) had no significant effect on the IFN alpha induced membrane potential changes, or on membrane viscosity changes as measured by flow cytometry and electron spin resonance spectrometry. In tissue culture, indomethacin did not alter the anti-proliferative effect of IFN alpha on Daudi cells. Indomethacin may influence IFN synthesis, but not it's antiproliferative actions. Relatively high doses of pentoxifylline slightly inhibited proliferation of Daudi cells and synergized with IFN alpha. Measurement of early biophysical changes induced by IFNs may represent a new screening method to rapidly explore certain types of IFN alpha potentiating agents.

Increasing interest is being focused on the role of protein kinase C (PKC) in the mode of action of interferons (IFNs). Here we report that IFN-alpha induced a transient translocation of PKC from the cytosol to the particulate fraction of U937 cells. In contrast, after IFN-gamma treatment, no significant change in PKC activity could be observed. IFN-induced changes in membrane potential were also examined by means of a potential sensitive oxonal dye and flow cytometry. Hyperpolarization was induced by both IFN-alpha and IFN-gamma. The protein kinase inhibitor H-7 blocked the hyperpolarization induced by IFN-alpha but not by IFN-gamma. These concordant results suggest that PKC is involved in the early signals of IFN-alpha but not of IFN-gamma in U937 cells.

Paragangliomas of the retroperitoneum arise from specialized neural crest cells distributed along the aorta in association with the sympathetic chain. In order to ascertain characteristic CT features of extraadrenal retroperitoneal paragangliomas to differentiate them from other retroperitoneal tumors, 31 discrete tumors and two cases of paragangliomatosis in 28 patients were reviewed retrospectively, and the CT features were correlated with clinical and pathologic findings. There were 16 men and 12 women. Average age was 37 years (range, 11-70 years). Twenty-four patients (86%) had hypertension. Of these, catecholamine levels were elevated in all 18 patients who had biochemical studies. Four patients (14%) had malignant paragangliomas. The discrete tumors were classified by location as suprarenal (26%), renal hilar (32%), or infrarenal (42%). Suprarenal paragangliomas could not be distinguished from the ipsilateral adrenal gland on CT. The average size of functional tumors was smaller (7.0 cm) than that of nonfunctional tumors (12.0 cm), but the sizes of the two groups overlapped. Smaller tumors were more likely to be homogeneous and have well-defined margins than were larger tumors. Our findings indicate that extraadrenal retroperitoneal paragangliomas are functionally active more often than previously reported and that they are readily detected by CT as soft-tissue masses closely associated with the entire length of the abdominal aorta. However, no CT feature was found that was unique for paraganglioma.

The effect of human recombinant DNA interferon-alpha type A (rIFN alpha A) on nuclear lipid biosynthesis and on in vitro nucleic acid synthesis was investigated in Daudi lymphoma cells sensitive (Daudi Is) or resistant (Daudi Ir) to the antiproliferative activity of this glycoprotein. In the Daudi Is studied at 90 min and up to 8 h, relative proportions of 3H-labeled nuclear lipids were reproducibly altered as compared to controls: phosphatidylcholine increased while phosphatidylserine and total neutral lipids decreased. These changes were not detected in parallel studies of the whole cell extracts. No significant changes in the profiles of nuclear lipids were observed in Daudi Ir. Decreased rates of alpha DNA polymerase and of RNA transcription were evident within 90 min in the Daudi Is nuclei but not in untreated controls or nuclei from rIFN alpha A-treated Daudi Ir cells, thus suggesting a possible relationship of the rapid alterations of nuclear lipid biosynthesis in Daudi Is cells to the rIFN alpha A antiproliferative activity.

A challenging aspect of lung carcinogenesis is the elucidation of the mechanisms which permit initiated bronchial epithelial cells to attain a growth advantage over normal bronchial epithelial cells, and subsequently evolve into a malignant phenotype. In this review, the effects of interactions between normal and transformed cells, and the potential role of representative extrinsic factors on cell-cell communication are discussed. Evidence is presented to show how cell injury and the effects of serum and calcium may affect morphology and communication, and tumor development. A large number of autocrine-paracrine factors (e.g., TGF beta, TGF alpha) are released by bronchial epithelial cells. These factors may inhibit or promote the proliferation of normal and transformed bronchial epithelial cells, respectively. The ability of certain injurious and tumor promoting agents (e.g., formaldehyde, TPA) to select for the transformed phenotype may involve selective cell injury, the induction of terminal differentiation and an inhibition of gap junction communication among normal BE cells.

Exposure of human lymphoblastoid cell cultures to leukocyte interferon initiated the formation of membranous tubuloreticular inclusions (TRI) within the endoplasmic reticulum or perinuclear envelope. In four different cell lines originating from patients with lymphocytic cancers (Daudi, Raji, H-SB2, and SB), this unique ultrastructural effect displayed a log-linear relationship to increasing doses of interferon-alpha and was dose and time dependent. TRI morphogenesis began within 12 hr in Daudi or Raji cells exposed continuously to 500 IU of leukocyte interferon/ml, but only at 20 hr after 2- to 4-hr pulse exposures to 50 to 100 IU/ml. The TRI accumulation, determined by thin-section counts, reached maximal levels of up to 16% of cell sections within 48 to 72 hr. Experiments with Raji cells indicated a decrease in TRI formation during successive cell divisions; detection of TRI after a pulse of interferon was enhanced when DNA replication was arrested. TRI morphogenesis appeared to be independent of several other known biological actions of interferons. It manifested later than the induction of 2'-5'-oligoadenylate synthetase activity which has been correlated with establishment of the antiviral state and occurred in cell lines, including DaudiKIFNR, which resist the growth-inhibitory effects of leukocyte interferon. Participation of new polypeptide synthesis was indicated by experiments with inhibitors of transcription (actinomycin D) or translation (cycloheximide): TRI morphogenesis was blocked when actinomycin D was added 4 hr after interferon and was reduced when cycloheximide was added for the interval of 13 to 23 hr after interferon.

Tissue biopsies and peripheral blood samples from 10 patients with the characteristic clinical features of acquired immunodeficiency syndrome (AIDS) were examined by electron microscopy and ultracytochemical myeloperoxidase technique. Abundant tubuloreticular inclusions (TRI) were detected within the endoplasmic reticulum of capillary endothelial cells, histiocytes, and lymphocytes in kidneys, small bowel, and lymph nodes, and lymphocytes and monocytes from peripheral blood. In general, TRI were found in the same type of cells and with conspicuous high frequency in our cases of AIDS as had been previously described in systemic lupus erythematosus (SLE). These findings indicate a morphologic link between these immunological disorders and the presence of TRI, raising the possibility of similar pathogenetic mechanisms.

Production of particles with the ultrastructural appearance of C-type virions persisted for at least 6 h in actinomycin D-treated cells infected with murine leukemia virus. This phenomenon occurred despite severe inhibition of viral RNA synthesis. Virus particles present in a 6-h harvest sedimented in sucrose gradients with the buoyant density characteristic of RNA tumor viruses (1.16 g/cm(3)) and exhibited high levels of reverse transcriptase activity in response to the exogenous template polyriboadenylic acid.oligo deoxythymidylic acid in the range of untreated controls. However, RNase-sensitive endogenous activity was only (1/5) the level found in controls. This observation correlated with a marked reduction in infectivity. Kinetic studies on the appearance of labeled RNA in banded virions revealed that within the first hour after addition of actinomycin D, particles contained 60 to 70S RNA and two low-molecular-weight RNA species corresponding to 8 and 4S RNA. After approximately 1 h of incubation with actinomycin D, 60 to 70S RNA could not be detected and 4S RNA was the predominant species. These findings suggest that murine leukemia virus particles assembled in the presence of actinomycin D are deficient in 60 to 70S viral RNA.

The morphology and occurrence of tubuloreticular and undulating membraneous structures (TRS and UMS) associated with the endoplasmic reticulum were examined under normal and pathologic conditions. It was concluded that TRS and UMS are cytoplasmic inclusions of similar type but of different organization. The occurrence of UMS in a human cell line derived from a carcinoma of the lung and in a transplantable chicken hepatoma derived from an MC-29 virus-induced liver tumour is described.

Two types of cytomembranous abnormalities were identified for the first time in liver biopsies from patients with chronic active type B hepatitis during treatment with recombinant alpha-interferon. Tubuloreticular inclusions were present in the hepatic endothelial cells, Kupffer cells and perisinusoidal cells of liver biopsies from both patients, and they were absent in liver biopsies obtained before treatment. Cylindrical confronting lamellae, having "test tube" or "ring-shape" forms were observed in the cytoplasm both of Kupffer cells and macrophages in the second liver biopsy of one of the patients. The findings suggest that interferon can be involved in the pathogenesis of both cytomembranous abnormalities, but that additional biological factors may play a role in formation of the cylindrical confronting lamellae.

Amygdalin, the gentiobioside derivative of mandelonitrile commonly referred to as Laetrile, is presently under intensive investigation as a potential cancer chemotherapeutic agent. Because of this interest, we investigated the activity of beta-glucosidases that cleave glucose from amygdalin and from prunasin (mandelonitrile monoglucoside) in tissues from germ-free rats and in normal and neoplastic human tissues. Rat and human small intestinal mucosa contain high levels of activity of glucosidases that act on both of these cyanogenic glucosides. Release of glucose from these compounds was not detected in any of the human neoplastic tissues examined in the present study. These observations are consistent with reports of cyanide toxicity through the oral use of amygdalin or prunasin and pose serious questions concerning the alleged tumoricidal effect of amygdalin.

Tubuloreticular inclusions (TRI) developed within the endoplasmic reticulum of peripheral blood mononuclear cells (PBMC) sampled from eight patients with chronic type B hepatitis during cycles of therapy with DNA-recombinant human alpha-interferon (rIFN alpha A). Each cycle of therapy consisted of a series of six intramuscular injections (triweekly) of a fixed dose of rIFN alpha A (from 18 to 68 X 10(6) IU/dose). In PBMC examined by transmission electron microscopy, TRI were absent prior to therapy and developed during therapy in all cases. Peak serum levels of alpha-interferon (320 to 960 IU/ml) were achieved within 12 hours. At 24 hours. TRI were detected in 0.5 to 6.5% of PBMC sections, and they persisted in 1.4 to 6.8% of sections examined at 48 hours. After five sequential interferon doses, TRI were observed in 1.6 to 9.8% of PBMC sections. TRI could no longer be detected at 5 to 16 days after cessation of rIFN alpha A, but they reappeared during subsequent cycles of therapy. Subpopulations of the PBMC with TRI were differentiated by immunoelectron microscopy utilizing a battery of anti-Leu monoclonal antibodies: surface markers of T cells, helper/inducer or cytotoxic/suppressor T cell subsets, natural killer cells, or B-cells were identified by direct or indirect procedures utilizing avidin and biotinylated peroxidase. In cases analyzed with multiple monoclonal antisera, TRI were expressed in all of the major PBMC subpopulations. Monocytes with TRI were demonstrated by the endogenous peroxidase reaction. TRI were not found in circulating polymorphonuclear granulocytes. Lymphocytes isolated from healthy donors and exposed to rIFN alpha A (100 IU/ml), for 48 to 72 hours in vitro, developed TRI in proportions of PBMC sections (2.3 to 8.4%) comparable to those observed in the interferon-treated patients. Stimulation of lymphocytes with concanavalin A, for 72 hours before rIFN alpha A exposure, enhanced formation of TRI which could then be found in T blasts. Stimulation of donor lymphocytes with Sendai virus, a potent inducer of alpha-interferon, also resulted in formation of TRI by 48 hours. This suggested that lymphocytotrophic virus infections could exercise a primary role in the natural pathogenesis of TRI.

A surface-adherent cell population developed spontaneously from Friend erythroleukemia cells (FLC745) which previously had grown continuously in suspension cultures. The adherent cells have been transferred through more than 60 passages and apparently represent a stable variant, herein designated FLC745-Ad. The cell size, chromosome complement, and tumorigenicity in DBA/2 mice were similar to the parental FLC745 line, although the FLC745-Ad cultures grew at a slightly slower rate and synthesized less hemoglobin in response to dimethyl sulfoxide. Production of C-type particles was abundant in both suspension and adherent cells, but the latter appeared to contain more intracisternal A-type particles. Scanning electron microscopy disclosed that most adherent cells were globoid and nonspreading. They attached to the substrate by a circumferential lamellar skirt of unusual breadth. Adhesion of the FLC745-Ad cells was not influenced by serum concentration, trypsinization, 5-bromodeoxyuridine, or N6, O2'-dibutyryl adenosine 3':5'-monophosphate. Treatment with cytochalasin B increased the cell size and the area of cell attachment.

A human agent of non-A, non-B hepatitis (Inoculum I) was transmitted to chimpanzees and alterations in liver and lymphocytes were studied by electron microscopy and by cytochemical techniques during the acute phase of the disease. Three types of cytoplasmic alterations, consisting of a membraneous and an amorphous part were observed in the hepatocytes. The density of the amorphous constituent decreased after treatment with pronase, but not after treatment with ribonuclease (RNase) or deoxyribonuclease (DNase). The wall of C-III, but not C-II had fibrils with a periodicity the contrast of which markedly increased after pronase treatment. Cytochemical data suggest that the inclusions (C-I-III) represent a cellular reaction to the infectious agent rather than the virus itself. Intranuclear vermicular inclusions (INI) were observed in hepatocytes and lymphocytes as well, mainly in degenerating cells. Tubuloreticular inclusions (TRS) did not appear in circulating lymphocytes during acute infection; however, they could be induced by human alpha interferon treatment in vitro. Increased numbers of lymphocytes with parallel tubular arrays (PTA) were noted at the peak of serum aminotransferase elevations. The latter two alterations (TRS and PTA) most likely represent immunologic reactions of the host to the infectious agent.

Nineteen specimens of peripheral blood mononuclear cells (PBMC) or biopsies of lymph nodes from patients with the acquired immunodeficiency syndrome (AIDS) were investigated ultrastructurally and by immunoelectron microscopy. Two main ultrastructural cytoplasmic inclusions were present in lymphoreticular cells: cylindrical confronting lamellae (CCL) and tubuloreticular inclusions (TRI). Furthermore their close ultrastructural association in the same cell was demonstrated for the first time. The percentage of PBMC sections bearing CCL was lower than the percentage of sections with TRI, except for one case of prodromal lymphadenopathy with risk factors for AIDS. The types of PBMC bearing inclusions were identified as T cells by immunoelectron microscopy. A relationship between the presence of TRI and plasma interferon levels is known but other unknown factor(s) are evidently involved in the morphogenesis of CCL.

The relationship between serum acid-labile alpha interferon and tubuloreticular inclusions within the cytoplasm of circulating lymphocytes was studied in 46 patients with systemic lupus erythematosus. Elevated levels of interferon (greater than or equal to 8 IU/ml) were found in 17 patients and lymphocyte inclusions in 35. The mean serum interferon concentration in patients with lymphocyte inclusions was significantly higher than in patients without inclusions (17.2 versus 2.4 IU/ml, p less than 0.01). Inclusions were found in 16 of 17 patients with elevated interferon and also in 19 of 29 patients without interferon (p = 0.026). In lupus, serum interferon appears to be a sufficient though not an essential marker for the presence of lymphocyte inclusions.

Human alpha interferons (IFN-a) cause a reorganization of internal cell membranes into tubuloreticular inclusions (TRI). Morphogenesis and cytochemistry indicate a pre-Golgi intracisternal origin from the endoplasmic reticulum. Clinically, TRI formation in human blood mononuclear cells correlates with systemic IFN-a treatment or with endogenous overproduction of IFN-a in viral or autoimmune diseases (e.g., rubella syndrome, AIDS, systemic lupus erythematosus). In vitro, TRI formation can be produced by treatment of Daudi lymphoblasts or vascular endothelial cells with IFN-a, and is blocked by actinomycin-D. In Daudi lymphoblasts or vascular endothelial cell cultures, TRI formation parallels induction of 2'-5' A synthetase, inhibition of thymidine kinase and growth inhibition; however, heavy water treatment of Daudi cells prevented TRI formation while induction of 2'-5' A synthetase and growth inhibition persisted. TRI formation was dissociated from IFN-a antiproliferative activity in a mutant clone of Daudi lymphoblasts. Decreased glycoprotein biosynthesis and increased phospholipid biosynthesis may accompany progressive TRI accumulation.

Anatomically widespread groups of extra-neural cells produce a spectrum of hormonal oligopeptides and monoamines comparable to those found in the central nervous system. Such cells express the capacity to cleave bioactive oligopeptides from polyprotein precursors, and the designation "neuroendocrine cells" has been widely adopted. Extra-neural cellular proliferations which exhibit the phenotypic manifestations of a neuroendocrine terminal differentiation also occur in a broad topographic distribution. Traditional tumor classifications reflected the wholistic concept of a "diffuse neuroendocrine system" in pathology. Newer results, however, including monoclonal antibody and in situ nucleic acid hybridization studies demonstrate that a variety of otherwise unrelated neoplasms can express the capacity to synthesize "ectopic" monoamines or hormonal oligopeptides. The molecular biological mechanisms which stimulate such cellular proliferations and regulate neuroendocrine expressions must be more diverse and complex than previously supposed. Critical alterations may involve multiple somatic mutations, abnormal gene transpositions or microenvironmental factors which could condition polyprotein gene transcription or post-transcriptional regulation. Accordingly, convergent patterns of neuroendocrine terminal differentiation in proliferative lesions and familial tumor complexes need not be pathogenetically homogenous. Molecular pathologic techniques now offer the potential to individualize diagnostic categories, and ultimately will facilitate more accurate clinical assessments of genetic risks, biologic progression, and therapeutic trials.