Molecular cloning of polyoma virus DNA in Escherichia coli: plasmid vector system.

A series of recombinant plasmids containing polyoma virus (PY) DNA were constructed, and their biological activity was evaluated in mice and in cultured mouse cells. While all of the recombinants studied contain the complete, potentially infectious viral DNA, in no case was the intact recombinant PY-plasmid DNA, or live Escherichia coli containing the recombinant plasmids, capable of inducing PY infection of mice, either by feeding or by parenteral injection.

Molecular cloning of polyoma virus DNA in Escherichia coli: oncogenicity testing in hamsters.

Inoculation of suckling hamsters with 2 x 10(8) live cells of Escherichia coli K12 strain chi1776, carrying the complete genome of polyoma virus in a recombinant plasmid, failed to induce tumors in any of 32 recipients. Also, lambda phage DNA and particles with a monomeric insert of polyoma DNA did not induce tumors. Purified recombinant plasmid DNA, as well as phage particles and DNA containing a head-to-tail dimer of polyoma DNA, showed a low degree of oncogenicity, comparable to that of polyoma DNA prepared from mouse cells. These findings support the previous conclusions, based on infectivity assays in mice, that propagation of polyoma virus DNA as a component of recombinant DNA molecules in E. coli K12 reduces its biologic activity many orders of magnitude relative to the virus itself.

Molecular cloning of polyoma virus DNA in Escherichia coli: lambda phage vector system.

The biological activity of recombinant phage and recombinant phage DNA containing monomeric or dimeric polyoma DNA inserts was examined in mice and cultured mouse cells. Recombinant preparations containing a single copy of viral DNA were invariably noninfectious; molecules containing a dimeric polyoma DNA insert were at least seven orders of magnitude less infectious than polyoma virions after parenteral inoculation. No infection was detected with any recombinant preparation after oral administration.

Secretion-mediated STAT3 activation promotes self-renewal of glioma stem-like cells during hypoxia.

High-grade gliomas (HGGs) include the most common and the most aggressive primary brain tumor of adults and children. Despite multimodality treatment, most high-grade gliomas eventually recur and are ultimately incurable. Several studies suggest that the initiation, progression, and recurrence of gliomas are driven, at least partly, by cancer stem-like cells. A defining characteristic of these cancer stem-like cells is their capacity to self-renew. We have identified a hypoxia-induced pathway that utilizes the Hypoxia Inducible Factor 1α (HIF-1α) transcription factor and the JAK1/2-STAT3 (Janus Kinase 1/2 - Signal Transducer and Activator of Transcription 3) axis to enhance the self-renewal of glioma stem-like cells. Hypoxia is a commonly found pathologic feature of HGGs. Under hypoxic conditions, HIF-1α levels are greatly increased in glioma stem-like cells. Increased HIF-1α activates the JAK1/2-STAT3 axis and enhances tumor stem-like cell self-renewal. Our data further demonstrate the importance of Vascular Endothelial Growth Factor (VEGF) secretion for this pathway of hypoxia-mediated self-renewal. Brefeldin A and EHT-1864, agents that significantly inhibit VEGF secretion, decreased stem cell self-renewal, inhibited tumor growth, and increased the survival of mice allografted with S100ß-v-erbB/p53-/- glioma stem-like cells. These agents also inhibit the expression of a hypoxia gene expression signature that is associated with decreased survival of HGG patients. These findings suggest that targeting the secretion of extracellular, autocrine/paracrine mediators of glioma stem-like cell self-renewal could potentially contribute to the treatment of HGGs.

Insulin-like growth factor II-mediated proliferation of human neuroblastoma.

Neuroblastoma is an embryonal tumor that typically arises in cells of the developing adrenal medulla. IGF-II mRNA is expressed at high levels in the adrenal cortex before birth but it is not detectable until after birth in the adrenal medulla. Neuroblastoma cell lines corresponding to early adrenal medullary precursors did not express IGF-II, although all three cell lines we tested were growth stimulated by IGF-II. Cell lines corresponding to more mature adrenal medullary cells expressed IGF-II, and one, SK-N-AS, grows by an IGF-II autocrine mechanism (J. Clin. Invest. 84:829-839) El-Badry, Romanus, Helman, Cooper, Rechler, and Israel. 1989. An examination of human neuroblastoma tumor tissues for IGF-II gene expression using in situ hybridization histochemistry revealed that IGF-II is expressed by tumor cells in only 5 of 21 neuroblastomas, but is detectable in cells of nonmalignant tissues including adrenal cortical cells, stromal fibroblasts, and eosinophils in all 21 tumors. These findings indicate that IGF-II may function as an autocrine growth factor for some neuroblastomas and as a paracrine growth factor for others. They suggest that the growth regulatory pathways utilized by neuroblastoma mimic those used in the precursor cell type from which individual tumors arise.

Chromogranin A expression in normal and malignant human tissues.

We used a recombinant cDNA probe for human chromogranin A to measure the expression of mRNA encoded by this gene in a variety of normal human tissues and tumor specimens using Northern blot and in situ hybridization analysis. With few exceptions, the expression of chromogranin A mRNA appears to be restricted to normal tissues and tumors of neuroendocrine lineage. However, we have detected mRNA expression of this gene in 1 of 14 cell lines and 2 of 13 tumor specimens of colon adenocarcinoma. The finding of chromogranin A expression in some colon carcinomas suggests that a previously unrecognized subgroup of these tumors has neuroendocrine features. The detection of this subgroup demonstrates the potential for improving tumor classification through the use of techniques and reagents developed by recombinant DNA technology.

Differential protooncogene expression characterizes histopathologically indistinguishable tumors of the peripheral nervous system.

We have found highly predictable patterns of protooncogene expression in cell lines and tumor tissue of neuroblastoma (NB), a tumor of the peripheral nervous system (PNS). These patterns make it possible to recognize two different genetically definable subgroups among histopathologically indistinguishable tumors. Additionally, we have identified a difference in neurotransmitter biosynthetic enzyme activity in these two subgroups of NB. The patterns of protooncogene expression and neurotransmitter biosynthetic enzymes suggests that these tumors arise in different cells of the PNS.

Autonomous growth of a human neuroblastoma cell line is mediated by insulin-like growth factor II.

Insulin-like growth factor II (IGF-II) mRNA was increased in two of eight neuroblastomas and in eight of eight pheochromocytomas, tumors of the adrenal medulla that occur in childhood and adulthood, respectively. RNA encoding the type I IGF receptor, the receptor thought to mediate the mitogenic effects of IGF-I and IGF-II, also was uniformly expressed in these cells. To assess the role of IGF-II in the growth of these tumor cells, we have used the SK-N-AS cultured neuroblastoma cell line, which can be continuously propagated in mitogen-free medium, as a model system. Our results strongly suggest that IGF-II, synthesized by SK-N-AS cells and acting through type I IGF receptors, contributes to the autonomous growth of this tumor cell line. (a) SK-N-AS cells synthesized large amounts of IGF-II RNA and secreted greater than 50 ng/ml of IGF-II (as determined by specific radioimmuno- and radioreceptor assays). Little, if any, IGF-I RNA or immunoreactive IGF-I were detected. (b) SK-N-AS cells possess type I IGF receptors. (c) Exogenous IGF-I and IGF-II stimulated DNA synthesis in SK-N-AS cells, and this stimulation was abolished by a blocking antibody to the type I IGF receptor. (d) This anti-receptor antibody also abolished the multiplication of SK-N-AS cells in the absence of added mitogens. We conclude that IGF-II is an autocrine growth factor for SK-N-AS cells and suggest that this mechanism may contribute to the growth of some adrenal medullary tumors.

Plasticity of neuroblastoma tumor cells to differentiate along a fetal adrenal ganglionic lineage predicts for improved patient survival.

We have recently presented a model of human adrenal medullary histogenesis that incorporates all neural crest-derived lineages (chromaffin, sustentacular, and ganglionic) known to compose this tissue. To determine if neuroblastomas correspond to the arrested maturation of embryonal adrenal medullary cells, we evaluated the expression of adrenal medullary developmental markers in 81 neuroblastoma tumors. We found that patterns of chromaffin-related gene expression in these tumors correlated exactly with the patterns observed during maturation of adrenal medullary cells (P2 < 10(-5). In a multivariate Cox proportional hazards analysis of developmental marker expression and other well-recognized prognostic variables, evidence of maturation along a fetal ganglionic lineage, as monitored by HNK-1 immunoreactivity (relative risk of 6.42, P2 = 0.0001), and age at diagnosis (relative risk of 5.05, P2 = 0.0042) were independent and significant prognostic indicators of patient survival. These studies demonstrate that neuroblastomas correspond to embryonal adrenal medullary cells arrested at recognizable stages during development, and that evidence of maturation along a fetal ganglionic lineage appears to have major importance in predicting patient survival.

Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.

Cells which are highly proliferative typically lack expression of differentiated, lineage-specific characteristics. Id2, a member of the helix-loop-helix (HLH) protein family known to inhibit cell differentiation, binds to the retinoblastoma protein (pRb) and abolishes its growth-suppressing activity. We found that Id2 but not Id1 or Id3 was able to bind in vitro not only pRb but also the related proteins p107 and p130. Also, an association between Id2 and p107 or p130 was observed in vivo in transiently transfected Saos-2 cells. In agreement with these results, expression of Id1 or Id3 did not affect the block of cell cycle progression mediated by pRb. Conversely, expression of Id2 specifically reversed the cell cycle arrest induced by each of the three members of the pRb family. Furthermore, the growth-suppressive activities of cyclin-dependent kinase inhibitors p16 and p21 were efficiently antagonized by high levels of Id2 but not by Id1 Id3. Consistent with the role of p16 as a selective inhibitor of pRb and pRb-related protein kinase activity, p16-imposed cell cycle arrest was completely abolished by Id2. Only a partial reversal of p21-induced growth suppression was observed, which correlated with the presence of a functional pRb. We also documented decreased levels of cyclin D1 protein and mRNA and the loss of cyclin D1-cdk4 complexes in cells constitutively expressing Id2. These data provide evidence for important Id2-mediated alterations in cell cycle components normally involved in the regulatory events of cell cycle progression, and they highlight a specific role for Id2 as an antagonist of multiple tumor suppressor proteins.

Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription.

Id2 promotes apoptosis by a novel mechanism independent of dimerization to basic helix-loop-helix factors.

Members of the helix-loop-helix (HLH) family of Id proteins have demonstrated roles in the regulation of differentiation and cell proliferation. Id proteins inhibit differentiation by HLH-mediated heterodimerization with basic HLH transcription factors. This blocks their sequence-specific binding to DNA and activation of target genes that are often expressed in a tissue-specific manner. Id proteins can also act as positive regulators of cell proliferation. The different mechanisms proposed for Id-mediated promotion of entry into S phase also involve HLH-mediated interactions affecting regulators of the G1/S transition. We have found that Id2 augments apoptosis in both interleukin-3 (IL-3)-dependent 32D.3 myeloid progenitors and U2OS osteosarcoma cells. We could not detect a similar activity for Id3. In contrast to the effects of Id2 on differentiation and cell proliferation, Id2-mediated apoptosis is independent of HLH-mediated dimerization. The ability of Id2 to promote cell death resides in its N-terminal region and is associated with the enhanced expression of a known component of the programmed cell death pathway, the proapoptotic gene BAX.

Pediatric oncology: model tumors of unparalleled import.

Molecular studies of tumors arising during childhood have provided insights important for our understanding of the genetic and cellular events that now seem likely to mediate the development of many different malignancies. Of particular interest have been recent studies using recombinant DNA technology to study the pressure genetic alterations now thought to be central features of oncogenesis. Oncogenes and recessive cancer genes, first recognized to be of clinical importance during the study of Burkitt's lymphoma and retinoblastoma, are now thought to play a role in the development of most, if not all, tumors. Studies to identify more effective approaches to cancer prevention, detection, staging, and treatment are now seeking to build upon an understanding of those genetic alterations. It can be expected that pediatric oncology will once again play a pivotal role as these studies mature into clinical trials.

Differentiation of neuroblastoma enhances Bcl-2 expression and induces alterations of apoptosis and drug resistance.

Recent evidence suggests that resistance to antineoplastic therapy may result from mutations in genes mediating the apoptotic response to DNA damage. To determine the effects of epigenetic changes on tumor responsiveness to cytotoxic agents inducing DNA damage, we examined the chemosensitivity of neuroblastoma (NB) after differentiation by retinoic acid (RA). Differentiation of the cell lines SH-SY5Y and SMS-KCNR by RA abolished the cytotoxic effects of adriamycin (Adr) and cisplatin. Chemoresistance was not the result of decreased proliferation induced by RA because: (a) growth arrest by nutrient deprivation did not affect sensitivity; (b) growth arrested NB cell lines, which did not differentiate, remained chemosensitive; and (c) RA concentrations which promoted differentiation without affecting growth, induced resistance. Apoptosis characterized NB cells responding to Adr, although differentiated SH-SY5Y did not apoptose and were resistant to Adr and cisplatin. Marked induction of bcl-2 in NB cells followed RA-induced differentiation, whereas in cell lines failing to differentiate, bcl-2 was not detected. Our data indicate that NB differentiation induces drug resistance after a loss of the apoptotic response to antineoplastic drugs and suggest that bcl-2 overexpression is an important mechanism of resistance in differentiated tumor cells.

Manganese superoxide dismutase expression in human central nervous system tumors.

Manganese superoxide dismutase (MnSOD) is a superoxide anion scavenger located in mitochondria. Increased expression of MnSOD can diminish oxygen radical-mediated injuries and the cytotoxic effects of tumor necrosis factor alpha, ionizing radiation, and certain chemotherapeutic agents. We used immunohistochemical staining to analyze 42 specimens of human brain tumors and 3 normal brain controls with a polyclonal antibody recognizing human MnSOD. We measured MnSOD in cerebrospinal fluid (CSF) from 14 patients with brain tumors and 7 control patients using an ELISA. Although MnSOD is not readily detected in normal brain, malignant central nervous system tumors, including tumors metastatic to the brain, displayed marked immunoreactivity to MnSOD intracellularly, in the extracellular matrix and in the tumor endothelial cells. Grade IV astrocytomas (glioblastomas), Grade III astrocytomas, and medulloblastomas were strongly immunoreactive, whereas Grade II astrocytomas had much less immunoreactivity. ELISA analysis of CSF samples from patients with malignant tumors also revealed high levels of MnSOD protein, up to 45-fold greater than the level of control CSF samples.

Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic death of bystander cells.

An emerging strategy for cancer gene therapy involves the transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene into tumor cells, rendering them susceptible to the cytotoxic effects of ganciclovir. The observation that HSV-tk-expressing cells can also induce cell death in neighboring cells, which do not express HSV-tk, has been called the bystander effect. Gap junction-mediated transfer of cytotoxic molecules to bystander cells may be an important mechanism of bystander cell death, although others have suggested a role for phagocytosis. In this study, we evaluated the mode of cell death in bystander cells. We detected apoptosis in bystander cells and found that bystander cell death could be inhibited by BCL2 expression. We determined that ganciclovir incubations for 10 h were sufficient to induce cell death in most bystander cells cocultured with HSV-tk-expressing cells. During this period, no phagocytosis was detected, although it was obvious at later stages.

Beta 2-microglobulin expression in human embryonal neuroblastoma reflects its developmental regulation.

Class I major histocompatibility complex (MHC) antigen expression in neuroblastoma may play a role in the oncogenicity of this embryonal tumor of childhood. Since N-myc amplification in neuroblastoma tumors is associated with rapid tumor progression (33) and N-myc decreases Class I MHC antigen expression in rat neuroblastoma cells (21), we quantitated levels of N-myc mRNA and Class I MHC cell surface antigens in a panel of 24 human neuroblastoma cell lines. We found that N-myc expression is not invariably associated with low levels of beta 2-microglobulin (B2M) and Class I MHC antigen expression. As we considered that Class I MHC antigens may be regulated in association with the differentiation stage of the neuroblastoma tumor, we examined the expression of B2M during development of the human adrenal medulla, the tissue of origin of most neuroblastomas. We found that B2M is a marker of differentiated adrenal medullary cells, expressed late during the third trimester of development. Moreover, using morphological and immunological criteria, we found that B2M is expressed in differentiated tumor cells. These data suggest that the expression of B2M in neuroblastoma is associated with the stage of differentiation of the tumor cell and not N-myc expression. Furthermore, these findings suggest that neuroblastomas may correspond to the arrested differentiation of adrenal neuroblasts at different stages of development.

Increased N-myc expression following progressive growth of human neuroblastoma.

Many human neuroblastoma tumors and cells in culture contain amplified N-myc DNA and this is associated with tumor stage. We have analyzed pairs of neuroblastoma cell cultures derived from two patients at the time of diagnosis and after tumor progression following the initiation of therapy. Cell cultures derived after progression have increased expression of N-myc RNA. In one pair this increase is associated with increased N-myc DNA amplification; in the other, amplification decreases and activation of N-myc is most likely the result of a regulatory change. Analysis of the pattern of DNA amplification in these cell cultures demonstrates additional changes that might be associated with tumor progression.

Expression of nitric oxide synthase in human central nervous system tumors.

The nitric oxide synthases (NOS) are a family of related enzymes which regulate the production of NO, a free radical gas implicated in a wide variety of biological processes. Vasodilation and increased tumor blood flow, increased vascular permeability, modulation of host tumoricidal activity, and free radical injury to tumor cells and adjacent normal tissues are pathophysiological features of malignant tumors that may be mediated by NO. We examined human brain tumors for three NOS isoforms and NADPH diaphrase, a histochemical marker of NOS activity in the brain. We detected increased expression of the brain and endothelial forms of NOS [NOS I and NOS II, respectively (C. Nathan and Q. Xie. Cell, 78: 915-919, 1994)] in astrocytic tumors, and the highest levels of expression was found in higher grade tumors. Each of these two isoforms was found in tumor cells and tumor endothelial cells. The macrophage isoform of NOS (NOS III) was less frequently detected and expressed at a lower level, predominantly in tumor endothelial cells. NADPH diaphorase staining for NOS activity paralleled this pattern of NOS expression. Western blot analysis of tumor tissues for these NOS isoforms confirmed these observations. Our data indicate that malignant central nervous system neoplasms express unexpectedly high levels of NOS and suggest that NO production may be associated with pathophysiological processes important to these tumors.

Developmental regulation of annexin II (Lipocortin 2) in human brain and expression in high grade glioma.

In experiments to identify molecules that might be important in the pathogenesis of glioblastoma multiforme, the most common malignant brain tumor, we found that annexin II (Lipocortin 2, p36), a likely second messenger in several different mitogenic pathways, was highly expressed in tumor tissue of glioblastoma multiforme (9 of 9) and highly anaplastic astrocytoma (2 of 6), but not in astrocytomas of lower pathological grade (0 of 6). We also detected high levels of annexin II expression in fetal brain during the period when radial glia proliferate, although annexin II expression was not detected in normal adult brain. These data demonstrate that annexin II expression is developmentally regulated in the human central nervous system and suggest that the early progenitor radial glia share important characteristics with highly malignant glial tumors.