Helen M. Pettersson

Staphylococcal enterotoxin H induces V alpha-specific expansion of T cells.

Staphylococcal enterotoxin H (SEH) is a bacterial superantigen secreted by Staphylococcus aureus. Superantigens are presented on the MHC class II and activate large amounts of T cells by cross-linking APC and T cells. In this study, RT-PCR was used to show that SEH stimulates human T cells via the Vα domain of TCR, in particular Vα10 (TRAV27), while no TCR Vβ-specific expansion was seen. This is in sharp contrast to all other studied bacterial superantigens, which are highly specific for TCR Vβ. It was further confirmed by flow cytometry that SEH stimulation does not alter the levels of certain TCR Vβ. In a functional assay addressing cross-reactivity, Vβ binding superantigens were found to form one group, whereas SEH has different properties that fit well with Vα reactivity. As SEH binds on top of MHC class II, an interaction between MHC and TCR upon SEH binding is not likely. This concludes that the specific expansion of TCR Vα is not due to contacts between MHC and TCR, instead we suggest that SEH directly interacts with the TCR Vα domain.

Arsenic trioxide is highly cytotoxic to small cell lung carcinoma cells

Small cell lung carcinoma (SCLC) is an extremely aggressive form of cancer and current treatment protocols are insufficient. SCLC have neuroendocrine characteristics and show phenotypical similarities to the childhood tumor neuroblastoma. As multidrug-resistant neuroblastoma cells are highly sensitive to arsenic trioxide (As2O3) in vitro and in vivo, we here studied the cytotoxic effects of As2O3 on SCLC cells. As2O3 induced pronounced cell death in SCLC cells at clinically relevant concentrations, and also at hypoxia. SCLC cells were more sensitive than non–SCLC cells to As2O3. Cell death was mainly due to necrosis, although apoptotic responses were also seen. A significant in vivo effect of As2O3 on SCLC growth was shown in a nude mice-xenograft model, although a fraction of the treated tumor-bearing animals did not respond. The nonresponding SCLC tumors differed in morphology and cell organization compared with treatment-responsive tumors, which in turn, showed decreased vascularization and higher expression of neuroendocrine markers compared with control tumors. Our results suggest a potential clinical application of As2O3 in SCLC therapy. In addition to cell death induction, antiangiogenic induction of differentiation may also be part of the in vivo effect of As2O3 on SCLC growth, as suggested by an increase in neuroendocrine markers in cultured cells. [Mol Cancer Ther 2009;8(1):160–70]

Multidrug-resistant neuroblastoma cells are responsive to arsenic trioxide at both normoxia and hypoxia

Despite intensive treatment, the outcome of high-risk neuroblastoma patients is poor with acquired multidrug resistance as an important cause. Previously, our group has shown that arsenic trioxide (As2O3) kills multidrug-resistant neuroblastoma cells in vitro and in vivo at clinically tolerable doses. Regions of tissue hypoxia often arise in aggressive solid tumors, and hypoxic tumors exhibit augmented invasiveness and metastatic ability in several malignancies. Furthermore, hypoxia may impair the treatment efficiency; therefore, we have studied the cytotoxic effect of As2O3 on neuroblastoma cells grown under normoxic as well as hypoxic (1% oxygen) conditions. At both normoxia and hypoxia, 2 and 4 μmol/L As2O3 induced evident cell death in the drug-sensitive SH-SY5Y and IMR-32 cells as well as in the multidrug-resistant SK-N-BE(2)c (with a mutated p53) and SK-N-FI cells after 72 hours of exposure. In contrast, the conventional chemotherapeutic drug etoposide showed lowered efficiency in hypoxic IMR-32 cells. In accordance with our previously published results, although not to the same extent as in their normoxic counterparts, Bax is proteolytically cleaved also in neuroblastoma cells exposed to As2O3 at hypoxia. This suggests that similar molecular mechanisms are involved in As2O3-induced neuroblastoma cell death during hypoxia compared with normoxia. Together, our results support As2O3 as a potential candidate drug as a complement to conventional treatments for high-risk neuroblastoma patients and perhaps also for patients with other multidrug-resistant solid tumors.

Relation between smoking history and gene expression profiles in lung adenocarcinomas

BackgroundLung cancer is the worldwide leading cause of death from cancer. Tobacco usage is the major pathogenic factor, but all lung cancers are not attributable to smoking. Specifically, lung cancer in never-smokers has been suggested to represent a distinct disease entity compared to lung cancer arising in smokers due to differences in etiology, natural history and response to specific treatment regimes. However, the genetic aberrations that differ between smokers and never-smokers’ lung carcinomas remain to a large extent unclear.MethodsUnsupervised gene expression analysis of 39 primary lung adenocarcinomas was performed using Illumina HT-12 microarrays. Results from unsupervised analysis were validated in six external adenocarcinoma data sets (n=687), and six data sets comprising normal airway epithelial or normal lung tissue specimens (n=467). Supervised gene expression analysis between smokers and never-smokers were performed in seven adenocarcinoma data sets, and results validated in the six normal data sets.ResultsInitial unsupervised analysis of 39 adenocarcinomas identified two subgroups of which one harbored all never-smokers. A generated gene expression signature could subsequently identify never-smokers with 79-100% sensitivity in external adenocarcinoma data sets and with 76-88% sensitivity in the normal materials. A notable fraction of current/former smokers were grouped with never-smokers. Intriguingly, supervised analysis of never-smokers versus smokers in seven adenocarcinoma data sets generated similar results. Overlap in classification between the two approaches was high, indicating that both approaches identify a common set of samples from current/former smokers as potential never-smokers. The gene signature from unsupervised analysis included several genes implicated in lung tumorigenesis, immune-response associated pathways, genes previously associated with smoking, as well as marker genes for alveolar type II pneumocytes, while the best classifier from supervised analysis comprised genes strongly associated with proliferation, but also genes previously associated with smoking.ConclusionsBased on gene expression profiling, we demonstrate that never-smokers can be identified with high sensitivity in both tumor material and normal airway epithelial specimens. Our results indicate that tumors arising in never-smokers, together with a subset of tumors from smokers, represent a distinct entity of lung adenocarcinomas. Taken together, these analyses provide further insight into the transcriptional patterns occurring in lung adenocarcinoma stratified by smoking history.

Arsenic trioxide-induced neuroblastoma cell death is accompanied by proteolytic activation of nuclear Bax.

Arsenic trioxide (As2O3) is toxic to multidrug-resistant neuroblastoma cells in vivo and in vitro. In neuroblastoma, As2O3 does not exert its cell death-promoting effects via a classical apoptotic pathway. A death mechanism involving proteolytic cleavage of Bax to a p18 form seems to be of importance, because inhibition of Bax cleavage coincides with diminished cell death. As existing models of cell death implicate Bax in the intrinsic apoptotic pathway, triggering death after Bax translocation to the mitochondria, we investigated the cellular localization of p18 Bax by subcellular fractionation. After As2O3 treatment, p18 Bax was only present in nuclei-enriched, mitochondria-depleted fractions. Cytoplasmic p21 Bax levels decreased, whereas total (p21 and p18) nuclear Bax increased. Overexpressed p21 Bax localized to the cytoplasm and nuclei, whereas overexpressed p18 Bax localized to extra-nuclear structures only. The inability of overexpressed p18 Bax to locate to the nucleus, and the As2O3-induced reduction of p21 Bax in the cytosol, suggest an As2O3-induced mechanism where p18 Bax gets cleaved and ‘trapped’ in the nucleus. This model is strengthened by the observation that calpain, the protease responsible for p18 Bax generation, is present in the nuclei, and that nuclear calpain is induced by increasing As2O3 and Ca2+ levels.

Staphylococcal enterotoxin H contrasts closely related enterotoxins in species reactivity

Staphylococcus aureus enterotoxin H (SEH) belongs to the staphylococcal enterotoxin (SE) family of superantigens (SAgs). SEH has structural similarities to other SE; however, its biological properties are less well characterized. SEH binds with high affinity to human major histocompatibility complex (MHC) class II and exhibits strong mitogenic activity in human T cells, although it was found to be less potent than the related SEA. Surprisingly and in sharp contrast to related SEs, SEH did not possess superantigen activity in murine T cells and T cells from three investigated rat strains. However, SEH bound to a high extent to murine MHC class II expressing cells and when presented by these cells SEH stimulated human T cells to proliferate. Thus, SEH interacts with the murine MHC class II molecule in a functional manner. Notably, SEH had an inhibitory effect on murine SEA response, demonstrating that SEH interferes with the SEA interactions with murine cells. Despite this, murine T cells did not proliferate regardless of whether SEH was presented on human or murine MHC class II expressing cells. Consequently, SEH differs in species reactivity as compared to related SEs and lacks critical properties for T‐cell activation in mice. We propose that unlike other SEs, SEH does not interact with murine T cells since it is not recognized by murine T‐cell receptors.

HIF-2α Expression Is Suppressed in SCLC Cells, Which Survive in Moderate and Severe Hypoxia When HIF-1α Is Repressed.

Small cell lung carcinoma (SCLC) is extremely aggressive and frequently metastasizes widely in its early stage. Because tumor hypoxia is related to aggressive tumor behavior and the hypoxic adaptation of SCLC is poorly documented, we stained SCLC tumors arranged in a tissue microarray for hypoxia-inducible factor (HIF)-1α and HIF-2α proteins. We found an overall lack of HIF-2α protein expression, which was confirmed in large tumor sections. HIF-1α protein was strongly expressed in most tumors, frequently adjacent to necrotic regions. In concordance, cultured SCLC but not non-small cell lung carcinoma cells showed no or extremely low levels of HIF-2α mRNA and no HIF-2α protein at hypoxia. HIF-1α was stabilized after 4 hours at hypoxia, and its accumulation increased up to 96 hours. SCLC cells survived well and showed net proliferation and low cell death in modest (1% oxygen) and severe (0.1% oxygen) hypoxia. HIF-1α repression virtually did not influence cell death or viability despite reduced levels of hypoxia-inducible genes, such as BNIP3 and BNIP3L. At 1% oxygen no increased autophagy (LC3B-II activation) or NF-κB signaling were detected, whereas the unfolded protein response was activated at severe hypoxia. Our data indicate that HIFs are not exclusively required for SCLC cell survival at modest or severe hypoxia and that additional, yet uncharacterized, hypoxia-driven adaptation pathways may become activated.

Abstract 452: The hypoxic response is differentially regulated in small cell lung carcinoma (SCLC) cells as compared to non-SCLC cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Here we study the adaptive capacity of small cell lung carcinoma (SCLC) cells to hypoxia and the importance of activation of hypoxia-inducible factor (HIF) transcription factors in these cells. SCLC is an extremely aggressive tumor form and the 5-year survival rate for these patients is only approximately 9-15%. Regions of hypoxia (∼1% oxygen) often appear in solid tumors and are associated with aggressive tumor behaviour and poor outcome in several tumor forms. Tumor cells adapt to a hypoxic environment by changing the transcription of genes involved in a variety of processes, like angiogenesis, cell survival, and metabolism. This adaptive response is primarily regulated by the transcription factors HIF-1α and HIF-2α, which become stable at low oxygen levels. Studies using among others neuroblastoma, breast carcinoma and lung adenocarcinoma cells have demonstrated that HIF-1α primarily mediates the acute hypoxic response, whereas HIF-2α dominates during the more chronic phase of hypoxia. We investigated cell survival in a panel of SCLC cell lines cultured at different oxygen pressures. In these cells we also studied expression levels of well known hypoxia-driven genes using quantitative real-time PCR. Quantitative real-time PCR, Western blot and immunohistochemistry were used to characterise the expression of HIF transcription factors in SCLC cells as well as in SCLC specimens. In all experiments a panel of non-SCLC cells and neuroblastoma cells were included for comparison. Since tumor hypoxia correlates with poor prognosis it is important to elucidate how tumor cells adapt in response to hypoxia. Our results show that SCLC cells survive well at hypoxia, although the proliferation is somewhat reduced with lower oxygen pressure. Even though the SCLC cells have a high adaptive capacity, only a moderate up-regulation was observed for the investigated hypoxia-regulated genes at 1% oxygen. In addition, the expression patterns of HIF-1α and HIF-2α differ in SCLC as compared to non-SCLC, indicating that a “typical” adaptive response to hypoxia does not occur in SCLC cells. Based on these results a gene expression profile of hypoxic SCLC cells using microarray analyses are performed to further characterise the hypoxic adaptive response. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 452.

Abstract 2058: The adaptation of small cell lung carcinoma (SCLC) cells to hypoxia is partially hypoxia inducible factor (HIF)-independent

Small cell lung carcinoma (SCLC) is an extremely aggressive tumor that is widely metastasized in early stages of the disease and the 5-year survival rate for these patients is only approximately 9-15%. Here we study the adaptive response of SCLC cells to lower oxygen levels, since tumor hypoxia (∼1% oxygen) is related to aggressive tumor behaviour and poor outcome in several tumor forms. Areas of hypoxia are a common characteristic in solid tumors and in response to hypoxia tumor cells induce expression of several genes involved in a variety of processes. This adaptive response is primarily regulated by the transcription factors hypoxia inducible factor (HIF)-1α and HIF-2α, which become stable at low oxygen levels. Studies using among others lung adenocarcinoma, neuroblastoma and breast carcinoma cells have demonstrated that HIF-1α primarily mediates the acute hypoxic response, whereas HIF-2α dominates during the more chronic phase of hypoxia. We investigated the viability of SCLC cells cultured at hypoxia and characterized the expression levels of HIF transcription factors in SCLC cells and specimens using quantitative real-time PCR, western blot and immunohistochemistry. Expression levels of well known hypoxia-driven genes were studied and to further characterize the hypoxic adaptive response, a gene expression profile of hypoxic SCLC cells was performed using microarray analyses. In all experiments a panel of non-SCLC cells and neuroblastoma cells were included for comparison. Our results show that SCLC cells have a high adaptive capacity to hypoxia, even though a modest induction of well known hypoxia-driven genes is observed. Interestingly, the HIF-1α and HIF-2α are differentially regulated in SCLC cells as compared to non-SCLC. In SCLC cells, no HIF-2α protein is detectable whereas HIF-1α protein levels are sustained and increased up to 96 hours. In addition, knock-down of HIF-1α at 0.1% oxygen are not affecting the viability of the SCLC cells and our gene expression profile of hypoxic SCLC cells has identified genes important for SCLC cells survival at hypoxia. Our data suggest that SCLC cells have an adaptive response to hypoxia that is partially HIF-independent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2011-2058