The neutralising and stimulatory effects of antimicrobial peptide LL-37 in human gingival fibroblasts.

OBJECTIVES: To investigate the effects of LL-37, a broad spectrum antimicrobial peptide expressed in periodontal tissues, on human gingival fibroblast responsiveness to microbial challenge and to explore the direct effects of LL-37 on human gingival fibroblasts. DESIGN: The effect of LL-37 on bacterial lipopolysaccharide-induced expression of Interleukin (IL-6) and chemokine C-X-C motif ligand (CXCL) 8 was determined by enzyme linked immunosorbent assay (ELISA). LL-37's influence on bacterial lipopolysaccharide-induced IκBα degradation was investigated by western blot. DNA microarray analysis initially determined the direct effects of LL-37 on gene expression, these findings were subsequently confirmed by quantitative polymerase chain reaction and ELISA analysis of selected genes. RESULTS: Bacterial lipopolysaccharide-induced IL-6 and CXCL8 production by human gingival fibroblasts was significantly reduced in the presence of LL-37 at concentrations in the range of 1-10 µg/ml. LL-37 led to a reduction in lipopolysaccharide-induced IκBα degradation by Escherichia coli lipopolysaccharide and Porphyromonas gingivalis lipopolysaccharide (10 µg/ml). LL-37 (50 µg/ml) significantly altered the gene expression of 367 genes in human gingival fibroblasts by at least 2-fold. CXCL1, CXCL2, CXCL3, Interleukin-24 (IL-24), CXCL8, Chemokine (C-C motif) Ligand 2, and Suppressor of Cytokine Signalling 3 mRNA were significantly upregulated by LL-37. LL-37 also significantly stimulated expression of CXCL8, hepatocyte growth factor and CXCL1 at the protein level. CONCLUSION: LL-37 plays an important regulatory role in the immunomodulatory activity of gingival fibroblasts by inhibiting lipopolysaccharide -induced expression of inflammatory cytokines and directly stimulating the expression of an array of bioactive molecules involved in inflammation and repair.

Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes.

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

Genome-wide profiling of methylation identifies novel targets with aberrant hypermethylation and reduced expression in low-risk myelodysplastic syndromes.

Gene expression profiling signatures may be used to classify the subtypes of Myelodysplastic syndrome (MDS) patients. However, there are few reports on the global methylation status in MDS. The integration of genome-wide epigenetic regulatory marks with gene expression levels would provide additional information regarding the biological differences between MDS and healthy controls. Gene expression and methylation status were measured using high-density microarrays. A total of 552 differentially methylated CpG loci were identified as being present in low-risk MDS; hypermethylated genes were more frequent than hypomethylated genes. In addition, mRNA expression profiling identified 1005 genes that significantly differed between low-risk MDS and the control group. Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of the hypermethylated genes were underexpressed in low-risk MDS cases. Gene network analysis revealed molecular mechanisms associated with the low-risk MDS group, including altered apoptosis pathways. The two key apoptotic genes BCL2 and ETS1 were identified as silenced genes. In addition, the immune response and micro RNA biogenesis were affected by the hypermethylation and underexpression of IL27RA and DICER1. Our integrative analysis revealed that aberrant epigenetic regulation is a hallmark of low-risk MDS patients and could have a central role in these diseases.

Transcriptional dysregulation mediated by RUNX1-RUNX1T1 in normal human progenitor cells and in acute myeloid leukaemia.

The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.

Increased heterozygosity for MHC class II lineages in newborn males.

In plants, fungi and marine invertebrates, there are genetic compatibility systems to ensure diversity in the offspring. The importance of genetic compatibility in gametic union and selective abortion in vertebrate animals has also been appreciated recently. There have been suggestions that the major histocompatibility complex (HLA in humans) may be a compatibility system in vertebrates. HLA class II haplotypes often contain a second expressed DRB locus which can be either DRB3, DRB4 or DRB5. These encode the supertypical specificities and mark the ancestral lineages. The members of each lineage have related DNA sequences at the main class II locus HLA-DRB1. We analysed 415 newborns at all expressed DRB loci by PCR analysis to seek evidence for sex-specific prenatal selection events. While there was no significant change in heterozygosity rates between males and females at DRB1, the proportion of males carrying two DRB1 specificities from different ancestral lineages was significantly increased (53.7% in males vs 39.3% in females, P = 0.003). The genotypes consisting of phylogenetically most distinct ones, namely the DRB3 and DRB4 haplotypes, showed the most striking difference between sexes (P = 0.007). These results suggested a more favourable outcome for male concepti heterozygous for supertypical haplotypes. Heterozygosity for most divergent haplotypical families ensures the highest degree of functional heterozygosity at the main HLA class II locus DRB1 while increasing the likelihood of heterozygosity also at other MHC loci. Our observations agree with the previously reported heterozygote excess in male newborn rats and mice. Correlations between MHC class II heterozygosity and advertised male quality in deer and pheasant as well as increased reproductive success in MHC class II heterozygous male macaques are examples of postnatal benefits of heterozygosity in males that may be behind the development of prenatal selection mechanisms. The MHC-mediated prenatal selection of males may also be one of the selective events suggested by the very high primary (male-to-female) sex ratio at fertilization reaching close to unity at birth in humans. These results provide an appealing working hypothesis for further studies in humans and other vertebrates.

Elevated Bcr-Abl expression levels are sufficient for a haematopoietic cell line to acquire a drug-resistant phenotype.

A characteristic feature of chronic myeloid leukaemia (CML) is the inevitable advancement from a treatable chronic phase to a fatal, drug-resistant stage referred to as blast crisis. The molecular mechanisms responsible for this disease transition remain unknown. As increased expression of Bcr-Abl has been associated with blast crisis CML, we have established transfectants in 32D cells that express low and high levels of Bcr-Abl, and assessed their drug sensitivity. Cells with high Bcr-Abl expression levels are resistant to conventional cytotoxic drugs, and also require higher levels of STI571 (an inhibitor of Bcr-Abl), to induce cell death. Co-treatment with cytotoxic drugs and STI571 increased the sensitivity of the drug-resistant cells. Despite the drug-resistant phenotype, high Bcr-Abl levels concomitantly increased the expression of p53, p21, Bax and down-regulated Bcl-2. These cells maintain a survival advantage irrespective of a reduced proportion of cycling cells and the pro-apoptotic shift in gene expression. In addition, the level of Bcr-Abl expression (high or low) does not alter the growth factor independence and elevated Bcl-xL expression observed. Our study indicates that drug resistance can be primarily attained by increased Bcr-Abl expression, and highlights the potential of therapy which combines STI571 with conventional cytotoxic drugs.

Abnormalities of adherent layers grown from bone marrow of patients with myelodysplasia.

Myelodysplastic syndromes (MDS) are characterized by a clonal disorder of haemopoiesis with defective growth in vitro. The long-term culture system was used to examine aspects of stromal function in MDS patients. Primary long-term cultures of MDS bone marrow showed poor myelopoiesis with progenitors being detected for a median 3.5 weeks (n = 12) compared with 18 weeks in cultures of normal marrow (n = 10; P < 0.0001). The haemopoietic function of adherent layers was assessed in secondary co-cultures seeded with 5 x 10(6) cord blood mononuclear cells on irradiated normal (n = 27; aged 38-82 years) or MDS (n = 32; aged 41-86 years) adherent layers (> 60% confluent). The median myeloid progenitor number/cord blood co-culture was 135 in 5-week-old cultures with normal adherent layers and 22 in those with MDS layers (P < 0.0001). Myeloid colonies were detectable for a median 11 weeks with normal adherent layers and 6 weeks with MDS adherent layers (P < 0.0001); erythroid colonies were detectable for 7 weeks (normal) compared with 5 weeks (MDS) (P < 0.01). The differences in granulocyte-macrophage colony forming unit (CFU-GM) generation were not related to patient age. Cells from adherent layers of at least half of the primary normal (n = 48) and MDS (n = 26) long-term cultures expressed cytokines [interleukin (IL)-3, IL-1 beta, thrombopoietin (Tpo) and erythropoietin (Epo)] and receptors for retinoic acid (RAR alpha) [IL-2, IL-3, macrophage colony stimulating factor (M-CSF) (Fms) and Tpo (Mpl)]. Only IL-1 beta expression was reduced in week-5 MDS cultures compared with those from normal marrows (P < 0.05). There was also a highly significant decline in IL-1 beta expression in normal (but not MDS) adherent layers between week 5 and week 10. Thus, the adherent layers in cultures grown from MDS patients were haemopoietically defective and showed abnormal IL-1 beta expression.

An in vivo and in vitro comparison of the effects of b2-a2 and b3-a2 p210BCR-ABL splice variants on murine 32D cells.

The Philadelphia (Ph) chromosome, a characteristic cytogenetic marker of chronic myeloid leukaemia (CML), is caused by a reciprocal translocation juxtaposing the 3' region of the ABL gene onto the 5' region of the BCR gene. Due to conservation of the reading frame, but depending on the site of the breakpoint in the BCR gene, two alternatively spliced variants of the p210BCR-ABL mRNA (known as b2-a2 and b3-a2) are produced. To investigate whether there are any biological differences between these splice variants we have transfected the b3-a2 or b2-a2 cDNA into a murine myeloid cell line, 32D. We have also included the previously prepared 32Dp210 cell line (which expresses the b3-a2 transcript) in all of our comparisons. RT-PCR analysis indicated that transcription levels were comparable between the variants. Morphological examination of the cells expressing either of the BCR-ABL transcripts indicated that these cells were more mature with increased cytoplasm:nuclear ratios compared to the 32D parental and 32Dneo vector control cells. However, the 32Dp210 cells had a very different appearance from the other panel members and flow karyotyping indicated a clonal evolution and cytogenetic instability in these cells alone. At 10(6) and 10(7) cell doses all 32D cells expressing BCR-ABL caused ill health and tissue infiltration in SCID mice with such rapidity that statistical analysis was not informative. However, at the 10(5) and 10(4) dosage levels there were similar survival rates between mice injected with 32Db2-a2 or 32Db3-a2 while mice injected with 32Dp210 had a significantly shorter survival time. The study of this 32D cell line panel indicated that there were no overt differences in the biological properties conferred by the b3-a2 or b2-a2 transcripts to the 32D cells although these transcripts were able to confer in vitro and in vivo biological effects. This panel of BCR-ABL expressing 32D cells provides a useful model for CML disease progression studies.

High FUS/TLS expression in acute myeloid leukaemia samples.

Retinoic acid has the ability to induce differentiation in some myeloid leukaemia cell lines and has been used to induce remission in acute promyelocytic leukaemia patients. We have analysed changes in gene expression, by differential display, in HL60 cells exposed to all-trans retinoic acid (ATRA) for only 1 h. Only about 0.4% of the genes examined by this technique showed changes in expression level, and all four of the gene fragments identified were downregulated during the short 1 h exposure. Two of the fragments were novel, a third was MYC and the fourth was the FUS proto-oncogene. Northern analysis showed that FUS was downregulated within 1 h only during induced neutrophil differentiation but not at all during induced monocyte differentiation. Unlike the sensitive cell lines, ATRA-resistant cell lines did not show a downregulation of FUS over a 24 h period of exposure to ATRA. Using a semiquantitative PCR analysis, no difference in FUS levels was observed between ATRA-sensitive and -resistant cell lines. A similar analysis was carried out on primary acute myeloid leukaemia (AML), peripheral stem cell harvests (PBSC) and cord blood samples. The PBSC and cord blood samples had FUS levels that were similar or generally less than the cell lines. However, much higher levels were seen in 63% of the AML samples examined. The data presented are consistent with previous reports for a role for FUS in the promotion and maintenance of cellular proliferation.

Differential display as an approach to study differentiation and differentiation therapy in AML.

Different subgroups of acute myeloid leukemia (AML) can be defined by the specific non-random chromosomal translocation that is present within the abnormal cell types. In one type of AML, acute promyelocytic leukemia (APL), the block in the normal process of differentiation can be circumvented by the addition of a chemical inducer, in this case retinoic acid. This is due to the defect in APL affecting the retinoic acid receptor gene. This type of therapy has become known as differentiation therapy. However, most types of leukemia do not respond to the retinoic acid, and therefore methods of differentiation therapy need to be developed by targeting other genes involved in the leukemia process. This requires the molecular characterizations of the genes that are expressed during differentiation and in particular those genes that show a differential expression in inducer sensitive cells and those resistant to induced differentiation. Therefore, therapeutic agents could be developed to specifically target these genes. This article describes how the technique of differential display, as one of several possible methods of molecular screening, may allow the identification of genes which can be targeted to induce differentiation.

Inhibition of mitochondrial function in HL60 cells is associated with an increased apoptosis and expression of CD14.

The myelomonocytic cell line HL60 can be induced by a variety of chemical agents to differentiation to either neutrophils or monocytes. Examination of gene expression, by differential display, in cells induced to monocytes with 1alpha,25-dihydroxyvitamin D(3) or neutrophils with all-trans retinoic acid (ATRA) identified a number of clones with altered patterns of expression over the period of differentiation. One of these clones was the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) which showed a differential pattern of expression between the neutrophil and monocyte lineages. The potential of mitochondrial inhibitors to induce differentiation was investigated by treating the HL60 cells with either the NADH dehydrogenase inhibitor, Rotenone, the complex III inhibitor, Antimycin A, or the highly specific mitochondrial ATP-synthase inhibitor, Oligomycin. Although functional assays of differentiation did not produce any positive results, all the inhibitors resulted in a dramatic increase in CD14 expression at day 1, with CD38 markers not observed until day 3. The increased expression of CD14 was accompanied by a decrease in viability and all CD14 positive cells were also positive for Annexin V, a marker of apoptosis. These results suggest that inhibition of the components of the mitochondrial pathways may lead to the marking of some cells, via CD14, for cell death, whilst allowing commitment to differentiation to occur in the surviving population.

Unravelling an HLA-DR association in childhood acute lymphoblastic leukemia.

Genetic and environmental factors play an interactive role in the development of childhood acute lymphoblastic leukemia (ALL). Since the demonstration of a major histocompatibility complex (MHC) influence on mouse leukemia in 1964, an HLA association has been considered as a possible genetic risk factor. Despite extensive efforts, however, no strong evidence comparable to the H-2(k) influence on mouse leukemia has been shown. The number of negative serological studies resulted in a loss of interest and consequently, no molecular HLA-DR association study has been published to date. We reconsidered the HLA-DR association in childhood ALL in 114 patients from a single center and 325 local newborn controls by polymerase chain reaction (PCR) analysis of the HLA-DRB1/3/4/5 loci. With conventional analysis, there was a moderate allelic association with the most common allele in the HLA-DR53 group, HLA-DRB1*04, in the whole group that was stronger in males (P =.0005, odds ratio = 2.9). When the other expressed HLA-DRB loci were examined, homozygosity for HLA-DRB4*01, encoding the HLA-DR53 specificity, was increased in patients (21.1% v 8.3%; odds ratio = 2.9, P =.0005). Consideration of gender showed that all of these associations were reflections of a male-specific increase in homozygosity for HLA-DRB4*01 (32.8% v 4. 0%; odds ratio = 11.7, 95% confidence interval [CI] = 4.9 to 28.0; P = 3 x 10(-8)). This highly significant result provided the long-suspected evidence for the HLA-DR influence on the development of childhood ALL while confirming the recessive nature of the MHC influence on human leukemogenesis as in experimental models. The cross-reactivity between HLA-DR53 and H-2Ek, extensive mimicry of the immunodominant epitope of HLA-DR53 by several carcinogenic viruses, and the extra amount of DNA in the vicinity of the HLA-DRB4 gene argue for the case that HLA-DRB4*01 may be one of the genetic risk factors for childhood ALL.

Identification of a retinoic acid responsive aldoketoreductase expressed in HL60 leukaemic cells.

Neutrophil and monocyte differentiation can be induced in HL60 leukaemia cells by all-trans-retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3), respectively, whose differentiating effects can be enhanced by exposure to 'anti-inflammatory agents' and steroids. We have provided evidence that this potentiation is via inhibition of the activity of an enzyme of the aldoketoreductase (AKR) family, but had failed to identify expression of known AKRs in HL60 cells. In this study, we have identified a previously unclassified aldoketoreductase family member (termed HAKR e) that is expressed in HL60 cells. HAKR e is dramatically and transiently up-regulated in HL60 cells within 24 h of exposure to ATRA, further supporting the proposition that a member(s) of this family of enzymes play(s) a role in controlling cell growth and/or differentiation.

c-myc locus amplification and the acquisition of trisomy 8 in the evolution of chronic myeloid leukaemia.

The biological progression of chronic myeloid leukaemia is often associated with secondary cytogenetic abnormalities but the molecular mechanisms underlying this progression are poorly understood. This study explores the association of c-myc gene amplification with the progression of chronic myeloid leukaemia in fourteen individuals. Three of these cases showed amplification of c-myc during the course of their disease. Cytogenetic and molecular analysis of serial samples from some patients suggested the successive expansion of distinct clones of malignant cells. Our findings also suggest that trisomy 8 and locus amplification could represent alternative mechanisms for increasing c-myc gene dosage.

Identification of transcription factors expressed during ATRA-induced neutrophil differentiation of HL60 cells.

A recent clinical therapeutic initiative has been the use of chemical agents which induce the leukaemic cells to overcome their block in differentiation. In order to understand this block the cascade of molecular events needs to be characterized. Haemopoietic differentiation is ultimately controlled at the level of gene transcription which is mediated by an array of transcription factors. Many transcription factors contain similar structural protein sequences, and we have used an RT-PCR-based approach to isolate sequences, from transcription factor gene families which share similar domains. Degenerate primers corresponding to the TFIIIA zinc-finger consensus amino acid sequences and to the POU-homeodomain and POU-specific domain were used to amplify genes on the basis that they contained similarities in structural motifs shared within these families of transcription factors. A serum-independent HL60 cell line was induced towards the neutrophil lineage by treatment with all-trans retinoic acid (ATRA) for 24 h. CD38+ cells committed towards this lineage were enriched and a population of these cells treated with dihydroxyvitamin D3 to induce neutrophil maturation. RNA extracted from uninduced, ATRA-induced CD38+ cells, and vitamin D3 treated maturing cell cultures were amplified using the degenerate primers. PCR fragments were cloned, sequenced, clustered into homologous groups, and the group sequences searched on the GenBank database. The Oct 1 transcription factor, and a very close homologue, KIAA0144, was identified using the POU family primers. The zinc-finger primers identified three zinc-finger genes. The pattern of gene expression was suggested from the number of clones in each group at neutrophil commitment and maturation. The differential expression of the genes in the zinc finger and POU families will lead to a better understanding of the cascade of gene expression which occurs following ATRA-induced differentiation.

p53 mutations, methylation and genomic instability in the progression of chronic myeloid leukaemia.

In chronic myeloid leukaemia (CML), as with other tumour types, mutations of the p53 gene are associated with disease progression. Changes in regional methylation of DNA with CML tumour development have also been demonstrated. Methylation is one mechanism by which gene expression is controlled and the CpG sites, which are the targets of DNA methylation, are also the sites of a number of the mutations found in the p53 gene. Cells harbouring mutant p53 have been shown to accumulate further genomic and genetic aberrations and methylation which alters the conformation of DNA is also believed to play a role in genomic stability. There appears to be an interplay between p53 deregulation and changing methylation patterns with the progression of CML. The cause and effect of changes in both of these critical gene regulating, DNA repair and genomic stability factors and their deviation during the progression of CML will be discussed.