High concordance of genomic and cytogenetic aberrations between peripheral blood and bone marrow in myelodysplastic syndrome (MDS).

Bone marrow (BM) genetic abnormalities in myelodysplastic syndrome (MDS) have provided important biological and prognostic information; however, frequent BM sampling in older patients has been associated with significant morbidity. Utilizing single-nucleotide polymorphism array (SNP-A) and targeted gene sequencing (TGS) of 24 frequently mutated genes in MDS, we assessed the concordance of genetic abnormalities in BM and peripheral blood (PB) samples concurrently from 201 MDS patients. SNP-A karyotype in BM was abnormal in 108 (54%) and normal in 93 (46%) patients, with 95% (190/201) having an identical PB karyotype. The median copy number (CN) for deletions was significantly lower in BM (CN:1.4 (1-1.9)) than in PB (CN:1.5 (1-1.95), P<0.001). Using TGS, 71% (130/183) patients had BM somatic mutations with 95% (124/130) having identical mutations in PB. The mutant allele burden was lower in PB (median 27% (1-96%)) compared with BM (median 29% (1-100%); P=0.14) with no significant difference in the number, types of mutations or World Health Organization subtype. In all patients with discordant SNP (n=11) and mutation (n=6) profiles between BM and PB, shared abnormalities were always present irrespective of treatment status. Overall, 86% of patients had a clonal aberration with 95% having identical SNP-A karyotype and mutations in PB, thus enabling frequent assessment of response to treatment and disease evolution especially in patients with fibrotic or hypocellular marrows.

Human Gyrovirus Apoptin shows a similar subcellular distribution pattern and apoptosis induction as the chicken anaemia virus derived VP3/Apoptin.

The chicken anaemia virus-derived protein Apoptin/VP3 (CAV-Apoptin) has the important ability to induce tumour-selective apoptosis in a variety of human cancer cells. Recently the first human Gyrovirus (HGyV) was isolated from a human skin swab. It shows significant structural and organisational resemblance to CAV and encodes a homologue of CAV-Apoptin/VP3. Using overlapping primers we constructed a synthetic human Gyrovirus Apoptin (HGyV-Apoptin) fused to green fluorescent protein in order to compare its apoptotic function in various human cancer cell lines to CAV-Apoptin. HGyV-Apoptin displayed a similar subcellular expression pattern as observed for CAV-Apoptin, marked by translocation to the nucleus of cancer cells, although it is predominantly located in the cytosol of normal human cells. Furthermore, expression of either HGyV-Apoptin or CAV-Apoptin in several cancer cell lines triggered apoptosis at comparable levels. These findings indicate a potential anti-cancer role for HGyV-Apoptin.

Apoptin induces apoptosis by changing the equilibrium between the stability of TAp73 and ΔNp73 isoforms through ubiquitin ligase PIR2.

Apoptin, a protein derived from the chicken anaemia virus, induces cell death in various cancer cells but shows little or no cytotoxicity in normal cells. The mechanism of apoptin-induced cell death is currently unknown but it appears to induce apoptosis independent of p53 status. Here we show that p73, a p53 family member, is important in apoptin-induced apoptosis. In p53 deficient and/or mutated cells, apoptin induced the expression of TAp73 leading to the induction of apoptosis. Knockdown of p73 using siRNA resulted in a significant reduction in apoptin-induced cytotoxicity. The p53 and p73 pro-apoptotic target PUMA plays an important role in apoptin-induced cell death as knockdown of PUMA significantly reduced cell sensitivity to apoptin. Importantly, apoptin expression resulted in a marked increase in TAp73 protein stability. Investigation into the mechanisms of TAp73 stability showed that apoptin induced the expression of the ring finger domain ubiquitin ligase PIR2 which is involved in the degradation of the anti-apoptotic ∆Np73 isoform. Collectively, our results suggest a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2 resulting in the degradation of ∆Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.

PML involvement in the p73-mediated E1A-induced suppression of EGFR and induction of apoptosis in head and neck cancers.

Epidermal growth factor receptor (EGFR) tyrosine kinase is commonly overexpressed in human cancers; however, the cellular mechanisms regulating EGFR expression remain unclear. p53, p63 and p73 are transcription factors regulating many cellular targets involved in controlling the cell cycle and apoptosis. p53 activates EGFR expression, whereas TAp63 represses EGFR transcription. The involvement of p73 in the regulation of EGFR has not been reported. Here, a strong correlation between EGFR overexpression and increased levels of the oncogenic DeltaNp73 isoform in head and neck squamous cell carcinoma (HNSCC) cell lines was observed. Ectopic expression of TAp73, particularly TAp73beta, resulted in suppression of the EGFR promoter, significant downregulation of EGFR protein and efficient induction of cell death in all six EGFR-overexpressing HNSCC cell lines. EGFR overexpression from a heterologous LTR promoter protected lung cancer cells from TAp73beta-induced EGFR suppression and apoptosis. Expression of TAp73beta efficiently induced promyelocytic leukaemia (PML) protein expression and PML knockdown by shRNA attenuated the downregulation of EGFR and induction of apoptosis by p73 in HNSCC cells. Furthermore, PML was found to be important for E1A-induced suppression of EGFR and subsequent killing of HNSCC cells. Our data therefore suggest a novel pathway involving PML and p73 in the regulation of EGFR expression.

p400 function is required for the adenovirus E1A-mediated suppression of EGFR and tumour cell killing.

We have recently shown that E1A protein of human adenovirus downregulates epidermal growth factor receptor (EGFR) expression and induces apoptosis in head and neck (HNSCC) and lung cancer cells independently of their p53 status. E1A has five isoforms of which the major ones E1A12S and E1A13S regulate transcription of cellular genes by binding to transcriptional modulators such as pRB, CtBP, p300 and p400. In this study, we have identified E1A12S isoform to have the highest effect on EGFR suppression and induction of apoptosis in HNSCC cells. Similar to Ad5, E1A12S from human adenovirus types 2, 3, 9 and 12 suppressed EGFR, whereas E1A12S of adenovirus types 4 and 40 had no effect on EGFR expression. Using deletion mutants of E1A12S we have shown that interaction of E1A with p400, but not p300 or pRB, is required for EGFR suppression and apoptosis. Inhibition of p400 by short hairpin RNA confirmed that HNSCC cells with reduced p400 expression were less sensitive to E1A-induced suppression of EGFR and apoptosis. p300 function was shown to be dispensable, as cells expressing E1A mutants that are unable to bind p300, or p300 knockout cells, remained sensitive to E1A-induced apoptosis. In summary, this study identifies p400 as an important mediator of E1A-induced downregulation of EGFR and apoptosis.

Apoptin: specific killer of tumor cells?

In the early 1990s it was discovered that the VP3/Apoptin protein encoded by the Chicken Anemia virus (CAV) possesses an inherent ability to specifically kill cancer cells. Apoptin was found to be located in the cytoplasm of normal cells while in tumor cells it was localized mainly in the nucleus.(1) These differences in the localization pattern were suggested to be the main mechanism by which normal cells show resistance to Apoptin-mediated cell killing. Although the mechanism of action of Apoptin is presently unknown, it seems to function by the induction of programmed cell death (PCD) after translocation from the cytoplasm to the nucleus and arresting the cell cycle at G2/M, possibly by interfering with the cyclosome.(2) In addition, cancer specific phosphorylation of Threonine residue 108 has been suggested to be important for Apoptin's function to kill tumor cells.(3) In contrast to the large number of publications reporting that nuclear localization, induction of PCD and phosphorylation of Apoptin is restricted to cancer cells, several recent studies have shown that Apoptin has the ability to migrate to the nucleus and induce PCD in some of the normal cell lines tested. There is evidence that high protein expression levels as well as the cellular growth rate may influence Apoptin's ability to specifically kill tumor cells. Thus far both in vitro and in vivo studies indicate that Apoptin is a powerful apoptosis inducing protein with a promising prospective utility in cancer therapy. However, here we show that several recent findings contradict some of the earlier results on the tumor specificity of Apoptin, thus creating some controversy in the field. The aim of this article is to review the available data, some published and some unpublished, which either agree or contradict the reported "black and white" tumor cell specificity of Apoptin. Understanding what factors appear to influence its function should help to develop Apoptin into a potent anti-cancer agent.

Low-speed centrifugation of retroviral vectors absorbed to a particulate substrate: a highly effective means of enhancing retroviral titre.

For many gene therapy applications the effective titre of retroviral vectors is a limiting factor both in vitro and in vivo. Purification and concentration of retrovirus from packaging cell supernatant can overcome this problem. To this end we have investigated a novel procedure which involves complexing retrovirus to a dense and particulate substrate followed by a short low-speed centrifugation. The study reported here uses heat-killed, formaldehyde fixed Staphylococcus aureus (Pansorbin) absorbed to PG13 derived retrovirus. This complex was then used to harvest retrovirus from packaging cell supernatant: centrifugation and washing of this complex allows the retrovirus to be both purified and concentrated. This procedure increases the effective titre of retrovirus by up to 7500-fold after an only 200-fold reduction in volume. The affinity of Pansorbin for retrovirus allows concentration regardless of its encoded genes and makes this protocol applicable to other popular packaging cells and envelope proteins. Possible explanations for the marked increase in titre of concentrated virus and the mechanism governing the complexing of retrovirus to Pansorbin are discussed.

Local versus systemic interleukin-2: tumor formation by wild-type and B7-1-positive murine melanoma cells.

Modification of murine K1735 melanoma cells to express the immune costimulator B7-1 had no effect on tumor formation in syngeneic mice. In contrast, <40% of mice inoculated with K1735 cells modified to secrete murine interleukin-2 (IL-2) formed tumors, and no tumors formed when the K1735 cells coexpressed both murine IL-2 and B7-1. However, administration of systemic recombinant human IL-2 had no detectable effect on the formation of tumors by the B7-1-expressing K1735 cells. By contrast, admixtures of IL-2-secreting and B7-1-expressing K1735 cells formed fewer tumors than either cell type alone. Murine IL-2 was effective only when secreted locally, because the IL-2-secreting cells inoculated into the right flank did not affect the growth of the B7-1-expressing cells inoculated into the opposite flank.

Cloning and characterization of a retroviral plasmid, pCC1, for combination suicide gene therapy.

Introduction of the herpes simplex virus thymidine kinase (HSV-TK) gene into mammalian cells confers specific sensitivity to killing by the anti-herpes drug ganciclovir (GCV). This gene has therefore been used in a number of cancer gene therapy protocols as a therapeutic gene. However, the therapeutic efficacy of HSV-TK/GCV in cancer gene therapy experiments can be augmented by additional therapeutic genes. We have cloned a retroviral plasmid, pCC1, containing a fusion gene of HSV-TK and Sh-ble driven by an internal simian virus 40 early promoter. This gene encodes a fusion protein that confers GCV sensitivity and Zeocin resistance when introduced into mammalian cells. A multiple cloning site (MCS) allows the introduction of a second therapeutic gene under the transcriptional control of the Moloney murine leukemia virus 5' long terminal repeat. We have generated packaging cell lines electroporated with pCC1 or pCC1 rtIL-2 S (rat interleukin-2 gene cloned in the sense direction in the MCS), the supernatants of which transfer GCV sensitivity only, or both GCV sensitivity and rtIL-2 production, respectively to rat ovarian cancer cells. This plasmid may be useful for the study of combination suicide gene therapy strategies.

Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron.

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2 ). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.

Effect of costimulation and the microenvironment on antigen presentation by leukemic cells.

Costimulatory signals supplied by genetically modified tumor cells can enable T-cell recognition of tumor-associated antigens that were previously silent when presented by unmodified tumor cells. Although the mechanism of the CD80/CD28 costimulation has been studied extensively in the normal T-cell/antigen-presenting cell (APC) interactions, it is unclear how expression of CD80 by tumor cells mediates its effect. We demonstrate here that optimal CD80 expression on a leukemic cell enhances T-cell recognition of alloantigen primarily by lowering the level of T-cell receptor (TCR) stimulation required for activation. CD80 expression by leukemic cells leads to increased survival of activated T cells by inducing upregulation of the antiapoptotic protein BCL-2, but not BCL-X(L). The cytokine microenvironment in which T cells are activated is crucial in determining their differentiation and consequently the nature of the immune response generated. Many tumor cells produce immunosuppressive cytokines that may not favor the induction of cell-mediated immunity. In this study, the presence of CD80 on leukemic cells increased T-cell activation in vitro, but this did not result in the production of Th1 cytokines. We show that this is due to a leukemia-derived soluble factor that inhibits the production of Th1 cytokines. Optimal expression of a costimulatory molecule, therefore, enhances the ability of leukemic cells to present antigen by amplifying TCR signals, but the microenvironment generated by leukemic cells may suppress the immune response required for their eradication. Thus, strategies aimed at inducing antileukemic immunity by providing leukemic cells with costimulatory functions must ensure the presence of an appropriate microenvironment.

Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines.

One of the most important components of G1 checkpoint is the retinoblastoma protein (pRB110). The activity of pRB is regulated by its phosphorylation, which is mediated by genes such as cyclin D1 and p16/MTS1. All three genes have been shown to be commonly altered in human malignancies. We have screened a panel of 26 oral squamous cell carcinomas (OSCC), nine premalignant and three normal oral tissue samples as well as eight established OSCC cell lines for mutations in the p16/MTS1 gene. The expression of p16/MTS1, cyclin D1 and pRB110 was also studied in the same panel. We have found p16/MTS1 gene alterations in 5/26 (19%) primary tumours and 6/8 (75%) cell lines. Two primary tumours and five OSCC cell lines had p16/MTS1 point mutations and another three primary and one OSCC cell line contained partial gene deletions. Six of seven p16/MTS1 point mutations resulted in termination codons and the remaining mutation caused a frameshift. Western blot analysis showed absence of p16/MTS1 expression in 18/26 (69%) OSCC, 7/9 (78%) premalignant lesions and 8/8 cell lines. One cell line, H314, contained a frameshift mutation possibly resulting in a truncated p16/MTS1 protein. pRB was detected in 14/25 (56%) of OSCC but only 11/14 (78%) of these contained all or some hypophosphorylated (active) pRB. In premalignant samples, 6/8 (75%) displayed pRB, and all three normal samples and eight cell lines analysed contained RB protein. p16/MTS1 protein was undetectable in 10/11 (91%) OSCCs with positive pRB. Overexpression of cyclin D1 was observed in 9/22 (41%) OSCC, 3/9 (33%) premalignant and 8/8 (100%) of OSCC cell lines. Our data suggest p16/MTS1 mutations and loss of expression to be very common in oral cancer cell lines and less frequent in primary OSCC tumours. A different pattern of p16/MTS1 mutations was observed in OSCC compared to other cancers with all the detected p16/MTS1 mutations resulting in premature termination codons or a frameshift. The RB protein was expressed in about half (44%) of OSCCs and its expression inversely correlated with p16/MTS1 expression. In conclusion, we show that abnormalities of the RB pathway are a common mechanism of oral carcinogenesis.

Expression of a variant of CD28 on a subpopulation of human NK cells: implications for B7-mediated stimulation of NK cells.

The ability of NK cells to kill tumor cells is controlled by a balance between activating and inhibitory signals transduced by distinct receptors. In murine tumor models, the costimulatory molecule B7.1 not only acts as a positive trigger for NK-mediated cytotoxicity but can also overcome negative signaling transduced by MHC class I molecules. In this study, we have evaluated the potential of human B7.1-CD28 interaction as an activating trigger for human blood NK cells. Using multiparameter flow cytometric analysis and a panel of different CD28 mAbs, we show that human peripheral blood NK cells (defined by CD56+, CD16+, and CD3- surface expression) express the CD28 costimulatory receptor, with its detection totally dependent on the mAb used. In addition, the level of CD28 varies among individuals and on different NK cell lines, irrespective of CD28 steady-state mRNA levels. By performing Ab binding studies on T cells, our data strongly suggest that binding of two of the anti-CD28 Abs (clones 9.3 and CD28.2) is to a different epitope to that recognized by clones L293 and YTH913.12, which is perhaps modified in the CD28 molecule expressed by the NK cells. We also show that B7.1 enhances the NK-mediated lysis of NK-sensitive but not of NK-resistant tumor cells and that this increased lysis is dependent on CD28-B7 interactions as shown by the ability of Abs to block this lysis. Coculture of the B7.1-positive NK-sensitive cells also led to the activation of the NK cells, as determined by the expression of CD69, CD25, and HLA class II.

Modulation of Fos-mediated AP-1 transcription by the promyelocytic leukemia protein.

The growth and transformation suppressor function of promyelocytic leukemia (PML) protein are disrupted in acute promyelocytic leukemia (APL) as a result of its fusion to the RARalpha gene by t(15;17) translocation. There is significant sequence homology between the dimerization domain of PML and the Fos family of proteins, which imply that PML may be involved in AP-1 activity. Here we show that PML can cooperate with Fos to stimulate its AP-1-mediated transcriptional activity. Cotransfection of PML with GAL4/Fos strongly induced Fos-mediated activation of GAL4-responsive reporters, indicating a functional interaction between Fos and PML in vivo. Deletion analysis of Fos and PML demonstrated that the intact C-terminal domain of Fos (containing the dimerization domain), and the RING-finger, B1 box and nuclear localization domains of PML are involved in the cooperative activity of Fos and PML. Immunoprecipitation and electrophoretic mobility shift assay showed that PML is associated with the AP-1 complex. PMLRARalpha was also found to enhance the transcriptional activity of GAL4/Fos. The addition of retinoic acid abrogated the PMLRARalpha, but not PML-induced stimulation of GAL4/Fos activity in a dose-dependent manner. This study demonstrated that PML is involved in the AP-1 complex and can modulate Fos-mediated transcriptional activity, which may contribute to its growth suppressor function.

Enhanced immune costimulatory activity of primary acute myeloid leukaemia blasts after retrovirus-mediated gene transfer of B7.1.

Gene modification of malignant cells to express immune stimulators (cytokines and immune costimulators) has provided the basis for a novel form of immunotherapy. Using a MPSV-based retroviral vector with hygromycin resistance gene as a selectable marker, we have studied retrovirus-mediated gene transfer of an immune costimulator, B7.1, into primary human acute myeloid leukaemia (AML) cells and the subsequent induction of immune costimulatory function. AML blasts from 10 patients were transduced by co-culture for 48 h with or without haemopoietic growth factors (HGFs). In the absence of HGFs, transduction efficiency (TE), as judged by % B7.1 expressing cells, was low, varying from 0.3 to 8.2% (median 1.5%). Addition of HGFs increased the median TE 1.8-fold with stem cell factor alone and 2.6-fold with SCF, interleukin-3 and GM-CSF. Effects on cell cycling alone could not explain this difference, suggesting other factors such as virus binding and promoter activity, are also involved. CFU-AL assays indicated a higher transduction efficiency of clonogenic cells, which was not improved by growth factors. Limited duration of cell growth prevented significant expansion of transduced populations by culture in the presence of hygromycin. Although not increasing transduction efficiency, CD34 enrichment enhanced drug selection, by targeting cells with the greatest self-renewal capacity. Immunoselection of B7.1 expressing cells produced transduced populations with 30-60% expressing B7.1. In an allogeneic mixed leukaemic cell/T lymphocyte reaction (MLLR) transduced AML cells enriched by immunoselection were able to stimulate allogeneic T cells (CD4 and CD8 positive), which could be inhibited by a solubilised B7 receptor, CTLA4.Ig. Our results demonstrate that using a replication incompetent retroviral vector, it is possible to introduce the immune costimulator B7.1 into primary AML-blasts and by immunoselection, enrich the transduced cells, which may be used for subsequent administration as an autologous cellular vaccine.

Establishment of a novel radioligand assay using eukaryotically expressed cytochrome P4502D6 for the measurement of liver kidney microsomal type 1 antibody in patients with autoimmune hepatitis and hepatitis C virus infection.

BACKGROUND/AIMS: Liver kidney microsomal type 1 antibody (LKM1) is the diagnostic marker of autoimmune hepatitis (AIH) type 2 and is also found in patients with hepatitis C virus (HCV) infection. Cytochrome P4502D6 (CYP2D6) is the documented target antigen of LKM1 in AIH, but not in HCV infection. To compare the reactivity in the two conditions, we established a radioligand assay using eukaryotically expressed CYP2D6 as target. METHODS: A 1.2-kb human CYP2D6 cDNA was isolated from a human liver cDNA library and subcloned into an in vitro transcription vector pSP64 Poly(A). Recombinant CYP2D6 was then produced by in vitro transcription/translation, metabolically labelled with 35S methionine and used in the immunoprecipitation assay. Antibodies that bound radiolabelled CYP2D6 were immunoprecipitated and their levels assessed as cpm. Sera from 50 LKM1-positive patients (26 with AIH; 24 with HCV infection), 128 LKM1-negative patients and 57 normal controls were tested. RESULTS: Reactivity to 35S labelled CYP2D6 was observed in all LKM1-positive sera from patients with AIH and HCV infection, but in none of the controls. The cpm in both conditions were significantly higher than in normal controls (p<0.0001), and were correlated with the immunofluorescence titres of LKM1 (r 0.87, p<0.001 and r=0.64, p<0.001 for AIH and HCV infection, respectively). Reactivity to 35S labelled CYP2D6 was inhibited by addition of an excess of eukaryotically expressed CYP2D6. CONCLUSIONS: CYP2D6 is a major target antigen of both AIH and HCV infection. The novel radioligand assay is highly sensitive and specific.

Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors.

Previous studies have shown that expression of the immune co-stimulator B7.1 reduces the tumorigenicity of some, but not all, malignant cell lines. However, B7.1-expressing tumor cells are not very effective in inducing the rejection of established tumors. This may in part be due to induction of anergy in the potentially reactive T cells. Previous studies have shown that IL-2 can reverse the anergic state both in vitro and in vivo. Therefore, we have examined the effect of retrovirus-mediated delivery and expression of murine B7.1 and interleukin-2 on tumor formation and rejection of established MHC class I+/II- NC adenocarcinomas. Neither the expression of B7.1 nor IL-2 alone had a significant effect on NC tumorigenicity. In contrast, combined expression of B7.1 and IL-2 substantially decreased the tumorigenicity of these cells in the immunecompetent syngeneic hosts. T-cell depletion studies show this to be dependent primarily on the activation of CD4+ cells. Furthermore, distant subcutaneous injection of irradiated NC/IL-2/B7.1 can induce, much more effectively than NC/B7.1 or NC/IL-2, the rejection of small NC tumors, and prevent the recurrence of large surgically resected tumors. Together, these results suggest that tumor cells genetically modified to express B7.1 and IL-2 can induce the immune-mediated rejection of established class II- tumors by a mechanism involving CD4+ cells.