Jakob Stenman

Obatoclax, saliphenylhalamide and gemcitabine inhibit influenza A virus infection

Background: Novel options should be developed for treatment of IAV infections. Results: Obatoclax, saliphenylhalamide, and gemcitabine target host factors and inhibit IAV and several other viruses infections. Conclusion: These compounds represent potent antiviral agents. Significance: These compounds could be exploited in treatment of severe viral infections. Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

Relative levels of SCCA2 and scca1 mRNA in primary tumors predicts recurrent disease in squamous cell cancer of the head and neck

Squamous cell carcinoma antigen (SCCA) is widely used as a serum marker in cancers of the uterine cervix, the head and neck, lung and esophagus. Two isoforms of SCCA, deriving from 2 highly homologous serine proteinase inhibitor genes, are co‐expressed in normal and malignant squamous epithelium, but it is mainly the acidic isoform SCCA2 that is present in the circulation of cancer patients. We studied the relative levels of SCCA2 and SCCA1 mRNA in frozen sections of squamous cell carcinomas of the head and neck (SCCHN) in relation to disease recurrence, using a new reverse transcription‐polymerase chain reaction‐based technique for accurate quantitation of relative mRNA levels. Primary tumors from 30 SCCHN patients, recurrent tumors from 11 patients and normal epithelium from 16 controls were examined. In patients responding to initial therapy (n = 26), an elevated SCCA2/SCCA1 mRNA ratio in the primary tumor predicted recurrence independent of clinical stage (p = 0.011). The relative risk of developing a recurrence was 7.2 (CI 1.2–13.3) in patients with elevated vs. normal SCCA2/SCCA1 mRNA ratios. We demonstrate that subtle differences in expression levels of the SCCA genes are reflected in the course of the SCCHN disease and may provide a target for molecular grading of SCCHN tumors. If this finding can be confirmed in a larger study the SCCA2/SCCA1 mRNA ratio in primary tumors could be useful for individual selection of treatment strategy for patients with head and neck cancer.

Accurate determination of relative messenger RNA levels by RT-PCR

The increasing focus on functional genomics spurs new techniques for accurately quantitating differences in mRNA levels.

Detection of squamous‐cell carcinoma antigen‐expressing tumour cells in blood by reverse transcriptase‐polymerase chain reaction in cancer of the uterine cervix

We used a reverse transcriptase‐polymerase chain reaction method for squamous‐cell carcinoma (SCC) antigen mRNA to detect circulating tumour cells in patients with carcinoma of the uterine cervix. The sensitivity of the method, as determined by cell spiking experiments, was 10 cultured A431 cells among 106 white blood cells. Circulating tumour cells were detected in 6 of 15 patients. In our control group of 24 women, SCC antigen mRNA was detected in 2 pregnant women at term. We followed up the patients for 24 months after sampling and evaluated the outcome. Three out of 6 patients positive for SCC antigen mRNA have relapsed. Additionally, 1 patient has developed breast cancer. In the group of 9 patients negative for SCC antigen mRNA there has been 1 relapse and 1 case of progression of disease. These results suggest that detection of SCC antigen mRNA in peripheral blood by RT‐PCR could be useful for staging and evaluation of prognosis in epidermoid carcinoma of the uterine cervix. Int. J. Cancer 74:75‐80.

Multiple Heat Pulses during PCR Extension Enabling Amplification of GC-Rich Sequences and Reducing Amplification Bias

PCR amplification over GC-rich and/or long repetitive sequences is challenging because of thermo-stable structures resulting from incomplete denaturation, reannealing, and self-annealing of target sequences. These structures block the DNA polymerase during the extension step, leading to formation of incomplete extension products and favoring amplification of nonspecific products rather than specific ones. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to temporarily destabilize such blocking structures, in order to enhance DNA polymerase extension over GC-rich sequences. With this novel type of protocol, we were able to amplify all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes. The longest Fragile X expansion contained 940 CGG repeats, corresponding to about 2.8 kilo bases of 100% GC content. For the GNAQ and GP1BB genes, different length PCR products in the range of 700 bases to 2 kilobases could be amplified without addition of cosolvents. As this technique improves the balance of amplification efficiencies between GC-rich target sequences of different length, we were able to amplify all of the allelic expansions even in the presence of the unexpanded allele.

Overexpression of Human Chorionic Gonadotropin β Genes 3, 5 and 8 in Tumor Tissue and Urinary Cells of Bladder Cancer Patients

Objective: Human chorionic gonadotropin (hCG) is a marker of trophoblastic tumors, and the serum concentration of the free β-subunit (hCGβ) is an independent prognostic marker in several nontrophoblastic cancers. hCGβ is encoded by six genes, of which the type II genes (hCGβ 3/9, 5 and 8) are thought to be upregulated in relation to type I genes (hCGβ 6/7) in cancer. Method: We developed a novel quantitative RT-PCR method for the quantification of the relative expression levels of the two groups of hCGβ genes and analyzed 28 bladder tumors and 15 urine samples. Results: We found a higher relative expression level of type II genes in malignant compared with benign urothelia (p = 0.016) and in exfoliated urinary cells from cancer patients compared with those from benign controls (p = 0.026). The expression level was increasing with higher stage (p = 0.014) and grade (p = 0.001) and tended to be higher in relapsing tumors (p = 0.059). Conclusion: The increased hCGβ concentrations in body fluids of patients with aggressive bladder cancer may be due to overexpression of type II genes. Quantification of the relative mRNA expression levels of the hCGβ type I and II genes in urine cells should be further studied as a potential noninvasive tool for the diagnosis and follow-up of bladder cancer.

Detection of messenger RNA for the β-subunit of chorionic gonadotropin in urinary cells from patients with transitional cell carcinoma of the bladder by reverse transcription-polymerase chain reaction

We studied whether detection of messenger‐RNA (mRNA) for the beta‐subunit of chorionic gonadotropin (CGβ) in urinary cells from bladder cancer patients could be used as a marker of disease activity. Sixty‐eight urine samples from patients under follow‐up for bladder cancer and 23 samples from patients with other malignancies and non‐malignant surgical conditions, as well as 14 samples from healthy controls were analyzed. RNA was isolated from urinary cells collected by centrifugation. Reverse transcription‐polymerase chain reaction (RT‐PCR) was used to detect CGβ mRNA. The results were compared to those obtained by cystoscopy and urinary cytology. For comparison, we determined CG and CGβ in serum and urine and the core fragment of CGβ (CGβcf) in urine by immunofluorometric assays. CGβ mRNA was detected in 29 of 68 urine samples from patients with a history of bladder cancer, whereas all 14 samples from healthy controls tested negative. Elevated levels of CGβ were observed in serum in 18 of 45 bladder cancer patients, but the association with CGβ mRNA was weak. However, CGβ mRNA expression in the absence of detectable cancer also occurred in some conditions associated with cellular atypia such as urinary tract infection, instrumentation and certain therapies. There was a highly significant association between histologically verified transitional cell carcinoma of the bladder and CGβ mRNA in urine (p = 0.0014), implying CGβ mRNA expression in tumor tissue. We conclude that CGβ mRNA is a potential new marker for monitoring of bladder cancer. Further studies are needed to evaluate whether it provides independent clinical information. Int. J. Cancer (Pred. Oncol.) 84:304–308, 1999.

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Technique for strand-specific gene-expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription

Primer-independent cDNA synthesis during reverse transcription hinders quantitative analysis of bidirectional mRNA synthesis in eukaryotes as well as in cells infected with RNA viruses. We report a simple RT-PCR-based assay for strand-specific gene-expression analysis. By modifying the cDNA sequence during reverse transcription, the opposite strands of target sequences can be simultaneously detected by postamplification melting curve analysis and primer-initiated transcripts are readily distinguished from nonspecifically primed cDNA. We have utilized this technique to optimize the specificity of reverse transcription on a panel of 15 target genes. Primer-independent reverse transcription occurred for all target sequences when reverse transcription was performed at 42°C and accounted for 11%-57% of the final PCR amplification products. By raising the reaction temperature to 55°C, the specificity of reverse transcription could be increased without significant loss of sensitivity. We have also demonstrated the utility of this technique for analysis of (+) and (-) RNA synthesis of influenza A virus in infected cells. Thus, this technique represents a powerful tool for analysis of bidirectional RNA synthesis.

Immuno-modulating properties of saliphenylhalamide, SNS-032, obatoclax, and gemcitabine.

Influenza A viruses (IAVs) impact the public health and global economy by causing yearly epidemics and occasional pandemics. Several anti-IAV drugs are available and many are in development. However, the question remains which of these antiviral agents may allow activation of immune responses and protect patients against co- and re-infections. To answer to this question, we analysed immuno-modulating properties of the antivirals saliphenylhalamide (SaliPhe), SNS-032, obatoclax, and gemcitabine, and found that only gemcitabine did not impair immune responses in infected cells. It also allowed activation of innate immune responses in lipopolysaccharide (LPS)- and interferon alpha (IFNα)-stimulated macrophages. Moreover, immuno-mediators produced by gemcitabine-treated IAV-infected macrophages were able to prime immune responses in non-infected cells. Thus, we identified an antiviral agent which might be beneficial for treatment of patients with severe viral infections.