Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFß Signaling via Activation of Calcineurin.

Control of ionic gradients is critical to maintain cellular homeostasis in both physiological and pathological conditions, but the role of ion channels in cancer cells has not been studied thoroughly. In this work we demonstrated that activity of the Kv11.1 potassium channel plays a vital role in controlling the migration of colon cancer cells by reversing the epithelial-to-mesenchymal transition (EMT) into the mesenchymal-to-epithelial transition (MET). We discovered that pharmacological stimulation of the Kv11.1 channel with the activator molecule NS1643 produces a strong inhibition of colon cancer cell motility. In agreement with the reversal of EMT, NS1643 treatment leads to a depletion of mesenchymal markers such as SNAIL1, SLUG, TWIST, ZEB, N-cadherin, and c-Myc, while the epithelial marker E-cadherin was strongly upregulated. Investigating the mechanism linking Kv11.1 activity to reversal of EMT into MET revealed that stimulation of Kv11.1 produced a strong and fast inhibition of the TGFß signaling. Application of NS1643 resulted in de-phosphorylation of the TGFß downstream effectors R-SMADs by activation of the serine/threonine phosphatase PP2B (calcineurin). Consistent with the role of TGFß in controlling cancer stemness, NS1643 also produced a strong inhibition of NANOG, SOX2, and OCT4 while arresting the cell cycle in G0/G1. Our data demonstrate that activation of the Kv11.1 channel reprograms EMT into MET by inhibiting TGFß signaling, which results in inhibition of motility in colon cancer cells.

Notch-1-PTEN-ERK1/2 signaling axis promotes HER2+ breast cancer cell proliferation and stem cell survival.

Trastuzumab targets the HER2 receptor on breast cancer cells to attenuate HER2-driven tumor growth. However, resistance to trastuzumab-based therapy remains a major clinical problem for women with HER2+ breast cancer. Breast cancer stem cells (BCSCs) are suggested to be responsible for drug resistance and tumor recurrence. Notch signaling has been shown to promote BCSC survival and self-renewal. Trastuzumab-resistant cells have increased Notch-1 expression. Notch signaling drives cell proliferation in vitro and is required for tumor recurrence in vivo. We demonstrate herein a mechanism by which Notch-1 is required for trastuzumab resistance by repressing PTEN expression to contribute to activation of ERK1/2 signaling. Furthermore, Notch-1-mediated inhibition of PTEN is necessary for BCSC survival in vitro and in vivo. Inhibition of MEK1/2-ERK1/2 signaling in trastuzumab-resistant breast cancer cells mimics effects of Notch-1 knockdown on bulk cell proliferation and BCSC survival. These findings suggest that Notch-1 contributes to trastuzumab resistance by repressing PTEN and this may lead to hyperactivation of ERK1/2 signaling. Furthermore, high Notch-1 and low PTEN mRNA expression may predict poorer overall survival in women with breast cancer. Notch-1 protein expression predicts poorer survival in women with HER2+ breast cancer. These results support a potential future clinical trial combining anti-Notch-1 and anti-MEK/ERK therapy for trastuzumab-resistant breast cancer.

Deubiquitinase OTUD6B Isoforms Are Important Regulators of Growth and Proliferation.

Deubiquitinases (DUB) are increasingly linked to the regulation of fundamental processes in normal and cancer cells, including DNA replication and repair, programmed cell death, and oncogenes and tumor suppressor signaling. Here, evidence is presented that the deubiquitinase OTUD6B regulates protein synthesis in non-small cell lung cancer (NSCLC) cells, operating downstream from mTORC1. OTUD6B associates with the protein synthesis initiation complex and modifies components of the 48S preinitiation complex. The two main OTUD6B splicing isoforms seem to regulate protein synthesis in opposing fashions: the long OTUD6B-1 isoform is inhibitory, while the short OTUD6B-2 isoform stimulates protein synthesis. These properties affect NSCLC cell proliferation, because OTUD6B-1 represses DNA synthesis while OTUD6B-2 promotes it. Mutational analysis and downstream mediators suggest that the two OTUD6B isoforms modify different cellular targets. OTUD6B-2 influences the expression of cyclin D1 by promoting its translation while regulating (directly or indirectly) c-Myc protein stability. This phenomenon appears to have clinical relevance as NSCLC cells and human tumor specimens have a reduced OTUD6B-1/OTUD6B-2 mRNA ratio compared with normal samples. The global OTUD6B expression level does not change significantly between nonneoplastic and malignant tissues, suggesting that modifications of splicing factors during the process of transformation are responsible for this isoform switch. IMPLICATIONS: Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC. Mol Cancer Res; 15(2); 117-27. ©2016 AACR.

p53 Modulates Notch Signaling in MCF-7 Breast Cancer Cells by Associating With the Notch Transcriptional Complex Via MAML1.

p53 and Notch-1 play important roles in breast cancer biology. Notch-1 inhibits p53 activity in cervical and breast cancer cells. Conversely, p53 inhibits Notch activity in T-cells but stimulates it in human keratinocytes. Notch co-activator MAML1 binds p53 and functions as a p53 co-activator. We studied the regulation of Notch signaling by p53 in MCF-7 cells and normal human mammary epithelial cells (HMEC). Results show that overexpression of p53 or activation of endogenous p53 with Nutlin-3 inhibits Notch-dependent transcriptional activity and Notch target expression in a dose-dependent manner. This effect could be partially rescued by transfection of MAML1 but not p300. Standard and quantitative co-immunoprecipitation experiments readily detected a complex containing p53 and Notch-1 in MCF-7 cells. Formation of this complex was inhibited by dominant negative MAML1 (DN-MAML1) and stimulated by wild-type MAML1. Standard and quantitative far-Western experiments showed a complex including p53, Notch-1, and MAML1. Chromatin immunoprecipitation (ChIP) experiments showed that p53 can associate with Notch-dependent HEY1 promoter and this association is inhibited by DN-MAML1 and stimulated by wild-type MAML1. Our data support a model in which p53 associates with the Notch transcriptional complex (NTC) in a MAML1-dependent fashion, most likely through a p53-MAML1 interaction. In our cellular models, the effect of this association is to inhibit Notch-dependent transcription. Our data suggest that p53-null breast cancers may lack this Notch-modulatory mechanism, and that therapeutic strategies that activate wild-type p53 can indirectly cause inhibition of Notch transcriptional activity.

Non-small-cell lung carcinoma: role of the Notch signaling pathway.

Notch signaling plays a pivotal role during embryogenesis. It regulates three fundamental processes: lateral inhibition, boundary formation, and lineage specification. During post-natal life, Notch receptors and ligands control critical cell fate decisions both in compartments that are undergoing differentiation and in pluripotent progenitor cells. First recognized as a potent oncogene in certain lymphoblastic leukemias and mesothelium-derived tissue, the role of Notch signaling in epithelial, solid tumors has been far more controversial. The overall consequence of Notch signaling and which form of the Notch receptor drives malignancy in humans is deeply debated. Most likely, this is due to the high degree of context-dependent effects of Notch signaling. More recently, it has been discovered that Notch (especially Notch-1) can exert different, even opposite effects in the same tissue under differing microenvironmental conditions. Further complicating the understanding of Notch receptors is the recently discovered role for non-canonical Notch signaling. Additionally, the most frequent Notch signaling antagonists used in biological systems have been inhibitors of the transmembrane protease complex γ-secretase, which itself processes a plethora of class one transmembrane proteins and thus cannot be considered a Notch-specific upstream regulator. Here we review the available empirical evidence gathered in recent years concerning Notch receptors and ligands in non-small-cell lung carcinoma (NSCLC). Although an overview of the field reveals seemingly contradicting results, we propose that Notch signaling can be exploited as a therapeutic target in NSCLC and represents a promising complement to the current arsenal utilized to combat this malignancy, particularly in targeting NSCLC tissues under specific environmental conditions, such as hypoxia.

Depletion of Amyloid Precursor Protein (APP) causes G0 arrest in non-small cell lung cancer (NSCLC) cells.

We recently reported that Amyloid Precursor Protein (APP) regulates global protein synthesis in a variety of human dividing cells, including non-small cell lung cancer (NSCLC) cells. More specifically, APP depletion causes an increase of both cap- and IRES-dependent translation. Since growth and proliferation are tightly coupled processes, here, we asked what effects artificial downregulation of APP could have elicited in NSCLC cells proliferation. APP depletion caused a G0/G1 arrest through destabilization of the cyclin-C protein and reduced pRb phosphorylation at residues Ser802/811. siRNA to cyclin-C mirrored the cell cycle distribution observed when silencing APP. Cells arrested in G0/G1 (and with augmented global protein synthesis) increased their size and underwent a necrotic cell death due to cell membrane permeabilization. These phenotypes were reversed by overexpression of the APP C-terminal domain, indicating a novel role for APP in regulating early cell cycle entry decisions. It is seems that APP moderates the rate of protein synthesis before the cell clears growth factors- and nutrients-dependent checkpoint in mid G1. Our results raise questions on how such processes interact in the context of (at least) dividing NSCLC cells. The data presented here suggest that APP, although required for G0/G1 transitions, moderates the rate of protein synthesis before the cell fully commits to cell cycle progression following mechanisms, which seem additional to concurrent signals deriving from the PI3-K/Akt/mTORC-1 axis. APP appears to play a central role in regulating cell cycle entry with the rate of protein synthesis; and its loss-of-function causes cell size abnormalities and death.

Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis.

Multimodality Approaches to Treat Hypoxic Non-Small Cell Lung Cancer (NSCLC) Microenvironment.

We found both in vitro and in vivo that survival of NSCLC cells in a hypoxic microenvironment requires Notch-1 signaling. A hypoxic tumor environment represents a problem for NSCLC treatment because it plays a critical role in cancer resistance to chemotherapy, tumor recurrence, and metastasis. Here we targeted hypoxic tumor tissue in an orthotopic NSCLC model. We inhibited the Notch-1/IGF-1R/Akt-1 axis using 3 agents: a γ-secretase inhibitor or GSI (MRK-003), a fully humanized antibody against the human IGF-1R (MK-0646), and a pan-Akt inhibitor (MK-2206), alone or in various combinations including therapeutics currently in clinical use. All treatments but Akt inhibition significantly prolonged the median survival of mice compared with controls. GSI treatment caused specific cell death of hypoxic tumors. Tumors excised from mice displayed a significant reduction of markers of hypoxia. Moreover, GSI treatment caused reduced metastasis to the liver and brain. MK-0646 was not specific to a hypoxic tumor environment but substantially increased the median survival of treated mice compared with controls. NSCLC cells evaded MK-0646 treatment by specifically overactivating EGF-R both in vivo and in 5 cell lines in vitro. This phenomenon is achieved at the level of protein stability. MK-0646 treatment caused increased erlotinib sensitivity in NSCLC cells poorly responsive to it. Sequential treatment with MK-0646 followed by erlotinib prolonged median survival of mice significantly. When the 2 drugs were administered simultaneously, no survival benefit was observed, and this combination therapy proved less effective than MK-0646 used as single agent. Our data offer novel information that may provide insights for the planning of clinical trials in humans, likely for maintenance therapy of NSCLC patients.

Notch signaling in lung cancer.

Lung cancer is the leading cause of cancer-related deaths in the Western world. The lungs can be affected by a number of histologically diverse malignancies. Nonetheless, the vast majority of lung cancers are classified as non-small-cell lung cancer (NSCLC). Despite extensive research on different therapeutic regimens, the overall 5-year survival of patients diagnosed with NSCLC (all stages) is a dismal 15%. Although strongly correlated with tobacco smoke, there is an increasing NSCLC morbidity in individuals who have never smoked. The pattern of genetic lesions found in NSCLC derived from smokers and never-smokers appears to be different. This fact led to the hypothesis that different, still unidentified carcinogens are responsible for lung cancer onset in never-smokers. All the aforementioned considerations compel the scientific community to find novel therapeutic targets to fight such a deadly disease. In recent years critical pathways governing embryonic development have been increasingly linked to cancer. Here we will focus on the role of Notch signaling in lung cancer. Notch receptors' activity can be blocked through the use of different strategies, thus representing a promising alternative/complement to the arsenal of therapeutic strategies currently used to treat lung cancer.

Tissue Tropism of SV40 Transformation of Human Cells: Role of the Viral Regulatory Region and of Cellular Oncogenes.

SV40 has been detected prevalently in a limited panel of human tumors: mesothelioma, bone and brain tumors, and lymphoma. These are the same tumor types that are specifically induced by SV40 when injected into hamsters, a finding that has raised concerns about the possible pathogenic role of SV40 in humans. Two different SV40 isolates differing in the number of 72-bp elements in the virus regulatory region, archetypal SV40 (1ESV40), which contains one 72 bp, and nonarchetypal SV40 (wtSV40), which contains two 72 bp, have been detected in human tumors. 1ESV40 has been prevalently detected in brain tumors, with wtSV40 prevalently in mesothelioma. The apparent different cell tropism could be related to the virus (i.e., possibly to the number of 72-bp elements) and to different expression of cellular genes, known to play a critical role in SV40-mediated transformation of human cells, such as Notch-1 and c-Met. To test for possible differences in tissue tropism, we infected primary human mesothelial cells (HM) and primary human astrocytes (Ast) with 1ESV40 and with wtSV40 from 2 different SV40 strains, 776 and Baylor. All viruses transformed astrocytes; only wtSV40 transformed HM. Intracellular signaling of c-Met and Notch-1 was differently induced by these 2 viruses in HM and Ast. Differences in Notch-1 expression and signaling (i.e., downstream effectors, c-Myc, HEY-1, HES-1, and HEY-L) appeared to influence SV40-mediated transformation of primary astrocytes and mesothelial cells. Our results provide a biological rationale to the observation that 1ESV40 is prevalently detected in brain tumors and wtSV40 in mesotheliomas.

Opposite effects of Notch-1 and Notch-2 on mesothelioma cell survival under hypoxia are exerted through the Akt pathway.

Malignant mesothelioma (MM) is a cancer of the lining of the lungs, heart, and intestine and is known to respond poorly to chemotherapy. Here we show that malignant mesothelial cells have an elevated Notch signaling pathway compared with normal human mesothelial cells. We studied the role of Notch in MM under normoxic and hypoxic conditions, the latter condition best recapitulating the MM microenvironment. Genetic and chemical modulation of the Notch pathway indicated that MM cells are dependent on Notch signaling. More specifically, this signaling was Notch-1 dependent as the result of its negative transcriptional regulation on phosphatase and tensin homologue (PTEN), which led to activation of the prosurvival phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. Our study also provides evidence that whereas Notch-1 is elevated in the malignant setting, Notch-2 is diminished. This differential expression of the two Notch isoforms benefits cancer cell survival because reexpression of Notch-2 was toxic to MM cells. The mechanism of Notch-2 toxicity to MM cells countered that of Notch-1, as it was the result of positive transcriptional regulation of PTEN and inhibition of the PI3K/Akt/mTOR signaling pathway. These results provide new insight into the role of Notch in MM and suggest that Notch pathway inhibitors may be useful in the treatment of this deadly disease.

A novel mechanism of late gene silencing drives SV40 transformation of human mesothelial cells.

Suppression of the late gene expression, usually by integration of the viral DNA into the host genome, is a critical step in DNA tumor virus carcinogenesis. SV40 induces high rates of transformation in infected primary human mesothelial cells in tissue culture, leading to the formation of immortal cell lines (SV40-transformed human mesothelial cell lines, S-HML). The studies described here were designed to elucidate the unusual susceptibility of primary human mesothelial cells to SV40 carcinogenesis. We found that S-HML contained wild-type, mostly episomal SV40 DNA. In these cells, the early genes that code for the viral oncogenes are expressed; at the same time, the synthesis of the late genes, capsid proteins, is suppressed and S-HML are not lysed. Late gene suppression is achieved through the production of antisense RNA molecules. These antisense RNA molecules originate in the early region of the SV40 circular chromosome and proceed in antisense orientation into the late gene region, leading to the formation of highly unstable double-strand RNA, which is rapidly degraded. Our results reveal a novel biological mechanism responsible for the suppression of late viral gene products, an important step in viral carcinogenesis in humans.

The SV40 large T antigen-p53 complexes bind and activate the insulin-like growth factor-I promoter stimulating cell growth.

Inactivation of cellular p53 is a crucial step in carcinogenesis. Accordingly, p53 is inactivated in most human cancers by different mechanisms. In cells infected with DNA tumor viruses, p53 is bound to the viral tumor antigens (Tag). The current "dogma" views the Tag-p53 complexes as a way of sequestering and inactivating p53. Using primary human cells and SV40-transformed human cells, we show that in addition to inactivating p53 tumor suppressor activities, the Tag-p53 complex has growth stimulatory activities that are required for malignant cell growth. We found that in human cells, Tag-p53 complexes regulate transcription of the insulin-like growth factor I (IGF-I) gene by binding to the IGF-I promoter together with pRb and p300. Depletion of p53 leads to structural rearrangements of this multiprotein complex, resulting in IGF-I promoter transcriptional repression and growth arrest. Our data provide a novel mechanistic and biological interpretation of the p53-Tag complexes and of DNA tumor virus transformation in general. In the model we propose, p53 is not a passive inactive partner of Tag. Instead the p53-Tag complex promotes malignant cell growth through its ability to activate the IGF-I signaling pathway.

Oxygen concentration determines the biological effects of NOTCH-1 signaling in adenocarcinoma of the lung.

NOTCH signaling is an evolutionarily conserved signaling pathway that regulates cell fate during development and postnatal life. It has been increasingly linked to carcinogenesis, although its role in cancer seems to be highly context and tissue specific. Although NOTCH signaling is required for lung development, little is known about its role in lung cancer. In this study, we show that NOTCH signaling, as measured by the gamma-secretase cleavage product N(IC)-1, is active in both normal human and lung tumor samples; however, downstream NOTCH readouts (i.e., HES-1 and HES-5) are elevated in lung tumors. Levels of NOTCH signaling components in primary human lung cells reflect observations in tissue samples, yet lung tumor cell lines showed little NOTCH signaling. Because oxygen concentrations are important in normal lung physiology and lung tumors are hypoxic, the effect of low oxygen on these lung tumor cell lines was evaluated. We found that hypoxia dramatically elevates NOTCH signaling (especially NOTCH-1) in lung tumor cell lines and concomitantly sensitizes them to inhibition via small-molecule gamma-secretase inhibitors or NOTCH-1 RNA interference. gamma-Secretase inhibitor-induced apoptosis of lung tumor cells grown under hypoxic conditions could be rescued by reintroduction of active NOTCH-1. Our data strengthen the role of NOTCH in lung cancer and as a therapeutic target for the treatment of lung and other hypoxic tumor types.

Crocidolite asbestos and SV40 are cocarcinogens in human mesothelial cells and in causing mesothelioma in hamsters.

Only a fraction of subjects exposed to asbestos develop malignant mesothelioma (MM), suggesting that additional factors may render some individuals more susceptible. We tested the hypothesis that asbestos and Simian virus (SV40) are cocarcinogens. Asbestos and SV40 in combination had a costimulatory effect in inducing ERK1/2 phosphorylation and activator protein-1 (AP-1) activity in both primary Syrian hamster mesothelial cells (SHM) and primary human mesothelial cells (HM). Ap-1 activity caused the expression and activation of matrix metalloprotease (MMP)-1 and MMP-9, which in turn led to cell invasion. Experiments using siRNA and chemical inhibitors confirmed the specificity of these results. The same effects were observed in HM and SHM. Experiments in hamsters showed strong cocarcinogenesis between asbestos and SV40: SV40 did not cause MM, asbestos caused MM in 20% of hamsters, and asbestos and SV40 together caused MM in 90% of hamsters. Significantly lower amounts of asbestos were sufficient to cause MM in animals infected with SV40. Our results indicate that mineral fibers and viruses can be cocarcinogens and suggest that lower amounts of asbestos may be sufficient to cause MM in individuals infected with SV40.

Cellular and molecular parameters of mesothelioma.

Malignant mesotheliomas (MM) are neoplasms arising from mesothelial cells that line the body cavities, most commonly the pleural and peritoneal cavities. Although traditionally recognized as associated with occupational asbestos exposures, MMs can appear in individuals with no documented exposures to asbestos fibers, and emerging data suggest that genetic susceptibility and simian virus 40 (SV40) infections also facilitate the development of MMs. Both asbestos exposure and transfection of human mesothelial cells with SV40 large and small antigens (Tag, tag) cause genetic modifications and cell signaling events, most notably the induction of cell survival pathways and activation of receptors, and other proteins that favor the growth and establishment of MMs as well as their resistance to chemotherapy. Recent advances in high-throughput technologies documenting gene and protein expression in patients and animal models of MMs can now be validated in human MM tissue arrays. These have revealed expression profiles that allow more accurate diagnosis and prognosis of MMs. More importantly, serum proteomics has revealed two new candidates (osteopontin and serum mesothelin-related protein or SMRP) potentially useful in screening individuals for MMs. These mechanistic approaches offer new hope for early detection and treatment of these devastating tumors.

TNF-alpha inhibits asbestos-induced cytotoxicity via a NF-kappaB-dependent pathway, a possible mechanism for asbestos-induced oncogenesis.

Asbestos is the main cause of human malignant mesothelioma (MM). In vivo, macrophages phagocytize asbestos and, in response, release TNF-alpha and other cytokines that contribute to carcinogenesis through unknown mechanisms. In vitro, asbestos does not induce transformation of primary human mesothelial cells (HM); instead, asbestos is very cytotoxic to HM, causing extensive cell death. This finding raised an apparent paradox: How can asbestos cause MM if HM exposed to asbestos die? We found that asbestos induced the secretion of TNF-alpha and the expression of TNF-alpha receptor I in HM. Treatment of HM with TNF-alpha significantly reduced asbestos cytotoxicity. Through numerous technical approaches, including chemical inhibitors and small interfering RNA strategies, we demonstrate that, in HM, TNF-alpha activates NF-kappaB and that NF-kappaB activation leads to HM survival and resistance to the cytotoxic effects of asbestos. Our data show a critical role for TNF-alpha and NF-kappaB signaling in mediating HM responses to asbestos. TNF-alpha signaling through NF-kappaB-dependent mechanisms increases the percent of HM that survives asbestos exposure, thus increasing the pool of asbestos-damaged HM that are susceptible to malignant transformation. Cytogenetics supported this hypothesis, showing only rare, aberrant metaphases in HM exposed to asbestos and an increased mitotic rate with fewer irregular metaphases in HM exposed to both TNF-alpha and asbestos. Our findings provide a mechanistic rationale for the paradoxical inability of asbestos to transform HM in vitro, elucidate and underscore the role of TNF-alpha in asbestos pathogenesis in humans, and identify potential molecular targets for anti-MM prevention and therapy.

Some oral poliovirus vaccines were contaminated with infectious SV40 after 1961.

Some polio vaccines prepared from 1954 to 1961 were contaminated with infectious SV40. It has been assumed that all polio vaccines were SV40 free in the United States after 1961 and in other countries after 1962. Following a WHO requirement that was prompted by the detection of SV40 in some human tumors, we conducted a multilaboratory study to test for SV40 polio vaccines prepared after 1961. Vaccine samples from 13 countries and the WHO seed were initially tested by PCR. The possible presence of intact and/or infectious SV40 DNA in PCR-positive samples was tested by transfection and infection of permissive CV-1 cells. All results were verified by immunohistochemistry, cloning, and sequencing. All the vaccines were SV40 free, except for vaccines from a major eastern European manufacturer that contained infectious SV40. We determined that the procedure used by this manufacturer to inactivate SV40 in oral poliovirus vaccine seed stocks based on heat inactivation in the presence of MgCl2 did not completely inactivate SV40. These SV40-contaminated vaccines were produced from early 1960s to about 1978 and were used throughout the world. Our findings underscore the potential risks of using primary monkey cells for preparing poliovirus vaccines, because of the possible contamination with SV40 or other monkey viruses, and emphasize the importance of using well-characterized cell substrates that are free from adventitious agents. Moreover, our results indicate possible geographic differences in SV40 exposure and offer a possible explanation for the different percentage of SV40-positive tumors detected in some laboratories.