Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression

Inorganic arsenic, a human carcinogen, is enzymatically methylated for detoxication, consuming S-adenosyl-methionine (SAM) in the process. The fact that DNA methyltransferases (MeTases) require this same methyl donor suggests a role for methylation in arsenic carcinogenesis. Here we test the hypothesis that arsenic-induced initiation results from DNA hypomethylation caused by continuous methyl depletion. The hypothesis was tested by first inducing transformation in a rat liver epithelial cell line by chronic exposure to low levels of arsenic, as confirmed by the development of highly aggressive, malignant tumors after inoculation of cells into Nude mice. Global DNA hypomethylation occurred concurrently with malignant transformation and in the presence of depressed levels of S-adenosyl-methionine. Arsenic-induced DNA hypomethylation was a function of dose and exposure duration, and remained constant even after withdrawal of arsenic. Hyperexpressibility of the MT gene, a gene for which expression is clearly controlled by DNA methylation, was also detected in transformed cells. Acute arsenic or arsenic at nontransforming levels did not induce global hypomethylation of DNA. Whereas transcription of DNA MeTase was elevated, the MeTase enzymatic activity was reduced with arsenic transformation. Taken together, these results indicate arsenic can act as a carcinogen by inducing DNA hypomethylation, which in turn facilitates aberrant gene expression, and they constitute a tenable theory of mechanism in arsenic carcinogenesis.

Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-γ ligand troglitazone in patients with liposarcoma

Agonist ligands for the nuclear receptor peroxisome proliferator-activated receptor-γ have been shown to induce terminal differentiation of normal preadipocytes and human liposarcoma cells in vitro. Because the differentiation status of liposarcoma is predictive of clinical outcomes, modulation of the differentiation status of a tumor may favorably impact clinical behavior. We have conducted a clinical trial for treatment of patients with advanced liposarcoma by using the peroxisome proliferator-activated receptor-γ ligand troglitazone, in which extensive correlative laboratory studies of tumor differentiation were performed. We report here the results of three patients with intermediate to high-grade liposarcomas in whom troglitazone administration induced histologic and biochemical differentiation in vivo. Biopsies of tumors from each of these patients while on troglitazone demonstrated histologic evidence of extensive lipid accumulation by tumor cells and substantial increases in NMR-detectable tumor triglycerides compared with pretreatment biopsies. In addition, expression of several mRNA transcripts characteristic of differentiation in the adipocyte lineage was induced. There was also a marked reduction in immunohistochemical expression of Ki-67, a marker of cell proliferation. Together, these data indicate that terminal adipocytic differentiation was induced in these malignant tumors by troglitazone. These results indicate that lineage-appropriate differentiation can be induced pharmacologically in a human solid tumor.

Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2–13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 1½ years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.

Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.

PTEN controls tumor-induced angiogenesis

Mutations of the tumor suppressor PTEN, a phosphatase with specificity for 3-phosphorylated inositol phospholipids, accompany progression of brain tumors from benign to the most malignant forms. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of angiogenesis, a process termed the angiogenic switch. Therefore, we tested whether PTEN regulates tumor progression by modulating angiogenesis. U87MG glioma cells stably reconstituted with PTEN cDNA were tested for growth in a nude mouse orthotopic brain tumor model. We observed that the reconstitution of wild-type PTEN had no effect on in vitro proliferation but dramatically decreased tumor growth in vivo and prolonged survival in mice implanted intracranially with these tumor cells. PTEN reconstitution diminished phosphorylation of AKT within the PTEN-reconstituted tumor, induced thrombospondin 1 expression, and suppressed angiogenic activity. These effects were not observed in tumors reconstituted with a lipid phosphatase inactive G129E mutant of PTEN, a result that provides evidence that the lipid phosphatase activity of PTEN regulates the angiogenic response in vivo. These data provide evidence that PTEN regulates tumor-induced angiogenesis and the progression of gliomas to a malignant phenotype via the regulation of phosphoinositide-dependent signals.

Retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation

Human telomerase, a cellular reverse transcriptase (hTERT), is a nuclear ribonucleoprotein enzyme complex that catalyzes the synthesis and extension of telomeric DNA. This enzyme is specifically activated in most malignant tumors but is usually inactive in normal somatic cells, suggesting that telomerase plays an important role in cellular immortalization and tumorigenesis. Terminal maturation of tumor cells has been associated with the repression of telomerase activity. Using maturation-sensitive and -resistant NB4 cell lines, we analyzed the pattern of telomerase expression during the therapeutic treatment of acute promyelocytic leukemia (APL) by retinoids. Two pathways leading to the down-regulation of hTERT and telomerase activity were identified. The first pathway results in a rapid down-regulation of telomerase that is associated with retinoic acid receptor (RAR)-dependent maturation of NB4 cells. Furthermore, during NB4 cell maturation, obtained independently of RAR by retinoic X receptor (RXR)-specific agonists (rexinoids), no change in telomerase activity was observed, suggesting that hTERT regulation requires a specific signaling and occurs autonomously. A second pathway of hTERT regulation, identified in the RAR-responsive, maturation-resistant NB4-R1 cell line, results in a down-regulation of telomerase that develops slowly during two weeks of all-trans retinoic acid (ATRA) treatment. This pathway leads to telomere shortening, growth arrest, and cell death, all events that are overcome by ectopic expression of hTERT. These findings demonstrate a clear and full dissociation between the process of tumor cell maturation and the regulation of hTERT mRNA expression and telomerase activity by retinoids. We propose telomerase expression as an efficient and selective target of retinoids in the therapy of tumors.

Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.

Malignant transformation of early lymphoid progenitors in mice expressing an activated Blk tyrosine kinase

The intracellular signals governing cellular proliferation and developmental progression during lymphocyte development are incompletely understood. The tyrosine kinase Blk is expressed preferentially in the B lineage, but its function in B cell development has been largely unexplored. We have generated transgenic mice expressing constitutively active Blk [Blk(Y495F)] in the B and T lymphoid compartments. Expression of Blk(Y495F) in the B lineage at levels similar to that of endogenous Blk induced B lymphoid tumors of limited clonality, whose phenotypes are characteristic of B cell progenitors at the proB/preB-I to preB-II transition. Expression of constitutively active Blk in the T lineage resulted in the appearance of clonal, thymic lymphomas composed of intermediate single positive cells. Taken together, these results indicate that specific B and T cell progenitor subsets are preferentially susceptible to transformation by Blk(Y495F) and suggest a role for Blk in the control of proliferation during B cell development.

Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors

Endometrial stromal tumors are divided into three types: benign stromal nodules, endometrial stromal sarcomas, and undifferentiated endometrial sarcomas. A variety of cytogenetic abnormalities involving chromosome 7 have been reported in endometrial stromal sarcomas, including a recurrent t(7;17)(p15;q21). We have identified two zinc finger genes, which we have termed JAZF1 and JJAZ1, at the sites of the 7p15 and 17q21 breakpoints. Analyses of tumor RNA indicate that a JAZF1/JJAZ1 fusion is present in all types of endometrial stromal tumors; however, the fusion appears to be rarer among endometrial stromal sarcomas that would be considered high-grade according to certain classification schemes. These findings suggest that the less malignant endometrial stromal tumors may evolve toward more malignant types, but that some endometrial stromal sarcomas with relatively abundant mitotic activity may compose a biologically distinct group.

The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors.

Due to lack of effective therapy, primary brain tumors are the focus of intense investigation of novel experimental approaches that use vectors and recombinant viruses. Therapeutic approaches have been both indirect, whereby vectors are used, or direct to allow for direct cell killing by the introduced virus. Genetically engineered herpes simplex viruses are currently being evaluated as an experimental approach to eradicate malignant human gliomas. Initial studies with gamma (1)34.5 mutants, R3616 (from which both copies of the gamma (1)34.5 gene have been deleted) and R4009 (a construct with two stop codons inserted into the gamma (1)34.5 gene), have been assessed. In a syngeneic scid mouse intracranial tumor model, recombinant herpes simplex virus can be experimentally used for the treatment of brain tumors. These viruses and additional engineered viruses were subsequently tested in human glioma cells both in vitro and in vivo. Using a xenogeneic scid mouse intracranial glioma model, R4009 therapy of established tumors significantly prolonged survival. Most importantly, long-term survival was achieved, with histologic evidence that R4009 eradicated intracranial tumors in this model. Furthermore, the opportunity to evaluate gamma (1)34.5 mutants that have enhanced oncolytic activity, e.g., R8309 where the carboxyl terminus of the gamma (1)34.5 gene has been replaced by the murine homologue, MyD116, are considered.

Malignant conversion of chemically transformed normal human cells.

Two structurally unrelated chemicals, aflatoxin B1 and propane sultone, transformed human foreskin cells to a stage of anchorage-independent growth. Isolation from agar and repopulation in monolayer culture of these transformed cells was followed by transfection with a cDNA library, which resulted in cells that exhibited an altered epithelioid morphology. Chemically transformed/nontransfected cells and transfected normal cells did not undergo a significant morphological change. These epithelioid-appearing, transfected cells, when inoculated into nude mice, form progressively growing tumors. The tumors are histopathologically interpreted as carcinomas. All of the first generation tumors in the surrogate hosts exhibited characteristic rates of growth similar to those of transplants of spontaneous human tumors. In the second generation of tumor xenografts, the progressively growing tumors derived from the transfected cells exhibited a more rapid rate of growth. Southern analysis and reverse transcription PCR confirmed that a 1.3-kb genetic element was integrated into the genome and was actively being transcribed. Examination of the metaphase chromosomes in normal human cells revealed that the genetic element responsible for this conversion was located at site 31-32 of the q arm of chromosome 7. The DNA sequence of this 1.3-kb genetic element contains a coding region for 79 amino acids and a long 3'-untranslated region and appears to be identical to CATR1.3 isolated from tumors produced by methyl methanesulfonate-converted, nontransplantable human tumor cells.

4-Hydroxylation of estrogens as marker of human mammary tumors.

Estrogen is a known risk factor in human breast cancer. In rodent models, estradiol has been shown to induce tumors in those tissues in which this hormone is predominantly converted to the catechol metabolite 4-hydroxyestradiol by a specific 4-hydroxylase enzyme, whereas tumors fail to develop in organs in which 2-hydroxylation predominates. We have now found that microsomes prepared from human mammary adenocarcinoma and fibroadenoma predominantly catalyze the metabolic 4-hydroxylation of estradiol (ratios of 4-hydroxyestradiol/2-hydroxyestradiol formation in adenocarcinoma and fibroadenoma, 3.8 and 3.7, respectively). In contrast, microsomes from normal tissue obtained either from breast cancer patients or from reduction mammoplasty operations expressed comparable estradiol 2- and 4-hydroxylase activities (corresponding ratios, 1.3 and 0.7, respectively). An elevated ratio of 4-/2-hydroxyestradiol formation in neoplastic mammary tissue may therefore provide a useful marker of benign or malignant breast tumors and may indicate a mechanistic role of 4-hydroxyestradiol in tumor development.

High frequency of inactivating mutations in the neurofibromatosis type 2 gene (NF2) in primary malignant mesotheliomas.

Malignant mesotheliomas (MMs) are aggressive tumors that develop most frequently in the pleura of patients exposed to asbestos. In contrast to many other cancers, relatively few molecular alterations have been described in MMs. The most frequent numerical cytogenetic abnormality in MMs is loss of chromosome 22. The neurofibromatosis type 2 gene (NF2) is a tumor suppressor gene assigned to chromosome 22q which plays an important role in the development of familial and spontaneous tumors of neuroectodermal origin. Although MMs have a different histogenic derivation, the frequent abnormalities of chromosome 22 warranted an investigation of the NF2 gene in these tumors. Both cDNAs from 15 MM cell lines and genomic DNAs from 7 matched primary tumors were analyzed for mutations within the NF2 coding region. NF2 mutations predicting either interstitial in-frame deletions or truncation of the NF2-encoded protein (merlin) were detected in eight cell lines (53%), six of which were confirmed in primary tumor DNAs. In two samples that showed NF2 gene transcript alterations, no genomic DNA mutations were detected, suggesting that aberrant splicing may constitute an additional mechanism for merlin inactivation. These findings implicate NF2 in the oncogenesis of primary MMs and provide evidence that this gene can be involved in the development of tumors other than nervous system neoplasms characteristic of the NF2 disorder. In addition, unlike NF2-related tumors, MM derives from the mesoderm; malignancies of this origin have not previously been associated with frequent alterations of the NF2 gene.

Failure of programmed cell death and differentiation as causes of tumors: some simple mathematical models.

Most models of tumorigenesis assume that the tumor grows by increased cell division. In these models, it is generally supposed that daughter cells behave as do their parents, and cell numbers have clear potential for exponential growth. We have constructed simple mathematical models of tumorigenesis through failure of programmed cell death (PCD) or differentiation. These models do not assume that descendant cells behave as their parents do. The models predict that exponential growth in cell numbers does sometimes occur, usually when stem cells fail to die or differentiate. At other times, exponential growth does not occur: instead, the number of cells in the population reaches a new, higher equilibrium. This behavior is predicted when fully differentiated cells fail to undergo PCD. When cells of intermediate differentiation fail to die or to differentiate further, the values of growth parameters determine whether growth is exponential or leads to a new equilibrium. The predictions of the model are sensitive to small differences in growth parameters. Failure of PCD and differentiation, leading to a new equilibrium number of cells, may explain many aspects of tumor behavior--for example, early premalignant lesions such as cervical intraepithelial neoplasia, the fact that some tumors very rarely become malignant, the observation of plateaux in the growth of some solid tumors, and, finally, long lag phases of growth until mutations arise that eventually result in exponential growth.

Changes in expression of putative antigens encoded by pigment genes in mouse melanomas at different stages of malignant progression.

Cutaneous melanomas of Tyr-SV40E transgenic mice (mice whose transgene consists of the tyrosinase promoter fused to the coding regions of simian virus 40 early genes) strikingly resemble human melanomas in their development and progression. Unlike human melanomas, the mouse tumors all arise in genetically identical individuals, thereby better enabling expression of specific genes to be characterized in relation to advancing malignancy. The products of pigment genes are of particular interest because peptides derived from these proteins have been reported to function as autoantigens with immunotherapeutic potential in some melanoma patients. However, the diminished pigmentation characteristic of many advanced melanomas raises the possibility that some of the relevant products may no longer be expressed in the most malignant cells. We have therefore investigated the contributions of several pigment genes in melanotic vs. relatively amelanotic components of primary and metastatic mouse melanomas. The analyses reveal marked differences within and among tumors in levels of mRNAs and proteins encoded by the wild-type alleles at the albino, brown, slaty, and silver loci. Tyrosinase (the protein encoded by the albino locus) was most often either absent or undetectable as melanization declined. The protein encoded by the slaty locus (tyrosinase-related protein 2) was the only one of those tested that was clearly present in all the tumor samples. These results suggest that sole reliance on targeting tyrosinase-based antigens might selectively favor survival of more malignant cells, whereas targeting the ensemble of the antigens tested might contribute toward a more inclusive and effective antimelanoma strategy.