P1 Phenotype Research Articles

Effect of proteins from the coelomic fluid of superficially wounded holothurians on the activity of phagocytes

Holothurians are among the most capable of regenerating animals. Their phagocytes are analogues of macrophages, but the mechanisms of their participation in wound healing have not been studied. The aim of the work is to determine the effect of individual protein components isolated from the coelomic fluid of holothurians with superficial damage to the body wall on the functional activity of the two types of phagocytes (P1 and P2) in holothurian Eupentacta fraudatrix. Protein components of the coelomic fluid of holothurians with superficial wounds of the body wall were analyzed and collected by gel permeation chromatography. We used proteins whose content changed significantly during the wound healing period in preliminary experiments. Two proteins and a peptide were added to phagocytes, isolated by gradient centrifugation, simultaneously with the thermostable toxin of Yersinia pseudotuberculosis and incubated for 24 h. The production of superoxide anion radical was determined by the reduction of nitroblue tetrazolium (NBT) using a colorimetric method. To assess the specificity of the proteins, bovine serum albumin (BSA) was used as an internal control. In one-day in vivo experiments, proteins were administered to wounded animals. Two proteins, when compared with the effect of BSA, did not show a specific effect on the production of reactive oxygen species in phagocytes in vitro. However, a comparison of the effect of the peptide on the oxidative activity and viability of phagocytes with those of BSA revealed the specificity of its action. The peptide reduced the oxidative activity of P1 phagocytes and increased it in P2 phagocytes in a direct concentration-dependent manner. One of the two proteins being administered to wounded holothurians caused a multidirectional concentration-dependent effect on the oxidative activity of the two types of phagocytes, with a predominant suppression of the P1 phenotype activity. The ability of protein components to cause a shift in the ratio of oxidative activity of the two types of phagocytes towards the preferential activation of P2 phagocytes, which are considered as functional analogues of M2 macrophages, suggests that they act as anti-inflammatory agents.

Application of a simplified PCR-SSP method to detect A4GALT*01 and A4GALT*02 typing among Thai blood donors.

An intronic A4GALT single nucleotide variant, rs5751348:G>T, P2 or A4GALT*02 allele has a lower level of the enzyme-encoding A4GALT transcripts than the P1 individuals. Here, we first develop and validate a simple inhouse PCR-SSP method to detect A4GALT*01 and A4GALT*02 alleles, and second, apply this method to compare the allele frequencies between Thai and other populations. The conventional test tube technique was used to detect the P1 antigen in 222 blood samples from Thai blood donors at Thammasat University Hospital. A PCR-SSP method was optimized and validated for reproducibility and specificity to identify these alleles and was subsequently tested on 1,840 DNA samples of unknown phenotypes obtained from central, northern and southern Thais. In addition, allele frequencies of central Thais were compared with those of other populations. In the tested cohort (n=222), P1 and P2 phenotypes were typed in 26.13 and 73.87% of donors, respectively. The developed PCR-SSP was successfully optimized, and the outcomes were consistent with those of serological phenotyping and DNA sequencing results, demonstrating its validity for predicting P1/P2 phenotype. For central, northern and southern Thais, the A4GALT*01 frequency was 0.1579 (430/2,724), 0.1183 (71/600), and 0.2575 (206/800), whereas the A4GALT*02 frequency was 0.8421 (2,294/2,724), 0.8817 (529/600), and 0.7425 (594/800), respectively. Their observed frequencies among central Thais significantly differed from those in other populations (p<0.05). Our study has successfully developed a simple, precise, and reliable method to genotype A4GALT*01 and A4GALT*02 using inhouse developed PCR-SSP for predicting P1/P2 status.

The Prevalence of Lewis, Lutheran, and P1 Antigens and Phenotypes in Southwestern Saudi Arabia

The Prevalence of Lewis, Lutheran, and P1 Antigens and Phenotypes in Southwestern Saudi Arabia

The P1PK blood group system: revisited and resolved

This update on the P1PK blood group system (Hellberg Å, Westman JS, Thuresson B, Olsson ML. P1PK: the blood group system that changed its name and expanded. Immunohematology 2013;29:25-33) provides recent findings concerning the P1PK blood group system that have both challenged and confirmed old theories. The glycosphingolipids can no longer be considered the sole carriers of the antigens in this system because the P1 antigen has been detected on human red blood cell glycoproteins. New indications suggest that P1Pk synthase activity truly depends on the DXD motif, and the genetic background and molecular mechanism behind the common P1 and P2 phenotypes were found to depend on transcriptional regulation. Transcription factors bind the P1 allele selectively to a motif around rs5751348 in a regulatory region of A4GALT, which enhances transcription of the gene. Nonetheless, unexplained differences in antigen expression between individuals remain.

P1PK: a blood group system with an identity crisis

The P1PK blood group system (ISBT no. 003) was previously called the P blood group system, and the number of included antigens has varied over time. The history of the system is complicated and sometimes confusing, since several changes to the nomenclature have been made. Furthermore, the association between the antigens and their genetic home has raised many questions, as well as the long‐standing enigma regarding the molecular mechanism underlying the common P1 and P2 phenotypes. Step by step, the biochemical and genetic basis underlying the antigens expressed in this system has been revealed. This review will start with the historical background and continue with the latest findings, answering some of the questions such as why individuals with p phenotype lack not only Pk and P expression, but also P1, and whether the P1 antigens exist on both glycolipids and glycoproteins on the human red blood cells.

Yersinia pseudotuberculosis Thermostable Toxin Dysregulates the Functional Activity of Two Types of Phagocytes in the Holothurian Eupentacta fraudatrix

The effect of a thermostable toxin of Yersinia pseudotuberculosis (in comparison with that of dexamethasone) on the functional activities of two types of phagocytes (P1 and P2) was studied in the holothurian Eupentacta fraudatrix. A high level of NO was shown to be a marker of intact P1 cells, while the high activity of arginase was a marker of P2 cells. The antioxidant defense in the P1 type was more pronounced than that of the P2 type. At the same time, the toxin inhibited the functional activity (generation of reactive oxygen species) of P1 phagocytes after 1 h of incubation and induced primarily the activity of P2 phagocytes, compared to that of P1 cells, after 24 h. In contrast to dexamethasone, which induced the transformation of the P1 phenotype into the P2 phenotype, the toxin promoted the mutual acquisition of the phenotype features by these two types of phagocytes. An analogy between the P1 and P2 phagocytes of the holothurian and M1 and M2 macrophages is discussed.

Single nucleotide polymorphisms in A4GALT spur extra products of the human Gb3/CD77 synthase and underlie the P1PK blood group system.

Contrary to the mainstream blood group systems, P1PK continues to puzzle and generate controversies over its molecular background. The P1PK system comprises three glycosphingolipid antigens: Pk, P1 and NOR, all synthesised by a glycosyltransferase called Gb3/CD77 synthase. The Pk antigen is present in most individuals, whereas P1 frequency is lesser and varies regionally, thus underlying two common phenotypes: P1, if the P1 antigen is present, and P2, when P1 is absent. Null and NOR phenotypes are extremely rare. To date, several single nucleotide polymorphisms (SNPs) have been proposed to predict the P1/P2 status, but it has not been clear how important they are in general and in relation to each other, nor has it been clear how synthesis of NOR affects the P1 phenotype. Here, we quantitatively analysed the phenotypes and A4GALT transcription in relation to the previously proposed SNPs in a sample of 109 individuals, and addressed potential P1 antigen level confounders, most notably the red cell membrane cholesterol content. While all the SNPs were associated with the P1/P2 blood type and rs5751348 was the most reliable, we found large differences in P1 level within groups defined by their genotype and substantial intercohort overlaps, which shows that the P1PK blood group system still eludes full understanding.

The differential expression of the blood group P1 -A4GALT and P2 -A4GALT alleles is stimulated by the transcription factor early growth response 1.

The P1 /P2 phenotypic polymorphism is one of the earliest blood groups discovered in humans. These blood groups have been connected to different levels of expression of the A4GALT gene in P1 and P2 red blood cells; however, the detailed molecular genetic mechanism that leads to these two phenotypes has not been established. After our previous identification of an association between the single-nucleotide polymorphisms (SNPs) rs2143918 and rs5751348 in A4GALT gene and the P1 /P2 phenotype, we conduct a survey of transcription factors that might connect these SNPs with the differential expression of the P1 -A4GALT and P2 -A4GALT alleles. An in silico analysis of potential transcription factor binding motifs within the polymorphic SNPs rs2143918 and rs5751348 genomic regions was performed, and this was followed by reporter assays examining the candidate transcription factors, gene expression profiling, electrophoretic mobility shift assays, and P1 -A4GALT and P2 -A4GALT allelic expression analysis. The results revealed that the differential binding of transcription factor early growth response 1 to the SNP rs5751348 genomic region with the different genotypes in the A4GALT gene leads to differential activation of P1 -A4GALT and P2 -A4GALT expression. The present investigation, together with our previous study (Lai et al., Transfusion 2014;54:3222-31), have elucidated the molecular genetic details associated with the P1 /P2 blood groups.

Identification of the Molecular and Genetic Basis of PX2, a Glycosphingolipid Blood Group Antigen Lacking on Globoside-deficient Erythrocytes

The x2 glycosphingolipid is expressed on erythrocytes from individuals of all common blood group phenotypes and elevated on cells of the rare P/P1/P(k)-negative p blood group phenotype. Globoside or P antigen is synthesized by UDP-N-acetylgalactosamine:globotriaosyl-ceramide 3-β-N-acetylgalactosaminyltransferase encoded by B3GALNT1. It is the most abundant non-acid glycosphingolipid on erythrocytes and displays the same terminal disaccharide, GalNAcβ3Gal, as x2. We encountered a patient with mutations in B3GALNT1 causing the rare P-deficient P1 (k) phenotype and whose pretransfusion plasma was unexpectedly incompatible with p erythrocytes. The same phenomenon was also noted in seven other unrelated P-deficient individuals. Thin-layer chromatography, mass spectrometry, and flow cytometry were used to show that the naturally occurring antibodies made by p individuals recognize x2 and sialylated forms of x2, whereas x2 is lacking on P-deficient erythrocytes. Overexpression of B3GALNT1 resulted in synthesis of both P and x2. Knockdown experiments with siRNA against B3GALNT1 diminished x2 levels. We conclude that x2 fulfills blood group criteria and is synthesized by UDP-N-acetylgalactosamine: globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase. Based on this linkage, we proposed that x2 joins P in the GLOB blood group system (ISBT 028) and is renamed PX2 (GLOB2). Thus, in the absence of a functional P synthase, neither P nor PX2 are formed. As a consequence, naturally occurring anti-P and anti-PX2 can be made. Until the clinical significance of anti-PX2 is known, we also recommend that rare P1 (k) or P2 (k) erythrocyte units are preferentially selected for transfusion to P(k) patients because p erythrocytes may pose a risk for hemolytic transfusion reactions due to their elevated PX2 levels.

A rare Pk phenotype caused by a 433 C>T mutation of the β-1,3-N-acetylgalactosyltransferase gene

To study the serological characteristics and molecular mechanism for a rare Pk phenotype of the P1Pk blood group system. The blood group of the proband was identified by serological techniques. The coding region and flanking intronic sequences of the β-1,3-N-acetylgalactosyltransferase gene (B3GALANT1) associated with the Pk phenotype were analyzed using polymerase chain reaction sequence-based typing. The proband was identified as having a rare Pk phenotype including anti-P in her serum. The blood group of her daughter and husband showed a P2 phenotype. The nucleotide sequences of the B3GALANT1 gene of her husband and two randomly-chosen individuals were the same as the reference sequence (GenBank AB050855). Nucleotide position 433 C>T homozygous mutation in the B3GALANT1 was found in the proband, which has resulted in a stop codon at amino acid position 145, which may produce a premature protein capable of decreasing or inhibiting the activity of the β -1,3-N-acetylgalactosyltransferase. The nucleotide position 433 C/T heterozygous in the B3GALANT1 was found in her daughter. The Pk phenotype resulted from 433 C>T mutation in the B3GALANT1 gene has been identified.

Molecular basis for an individual with rare p phenotype in P1Pk blood group system

To explore the molecular basis for an individual with rare p phenotype in the P1Pk blood group system. Erythrocyte blood group antigens and antibodies in serum were identified in the proband and five family members with a serological method. Coding regions and flanking untranslated regions of the α1,4-galactosyltransferase gene (A4GALT) encoding P1Pk antigens were amplified with polymerase chain reaction and directly sequenced. The haplotypes of A4GALT in the parents of the proband were also analyzed by cloning sequencing. The proband was found with a rare p phenotype with anti-Tja antibody in his serum by serological method. The other family members all had a common P2 phenotype. The results of DNA sequencing showed that a cytosine was inserted at nucleotide position 1026 to 1029 (1026_1029insC) of both alleles of the A4GALT gene in the proband. The mutation has caused a reading frame shift and formed a mutant protein by extending 92 amino acid residues. The other family members were either heterozygous for the insertion or of the wild type at above position. The 1026_1029insC mutation of the A4GALT gene is probably responsible for the p phenotype identified for the first time in Chinese population. The individual with the p phenotype possesses anti-Tja antibody.

Emergence of the P2 Phenotype in Pseudomonas aeruginosa PAO1 Strains Involves Various Mutations in mexT or mexF

We recently demonstrated that Pseudomonas aeruginosa PAO1 undergoes a pronounced phenotypic change when introduced into the intestines of rats during surgical injury. Recovered strains displayed a specific phenotype (termed the P2 phenotype) characterized by altered pyocyanin production, high collagenase activity, high swarming motility, low resistance to chloramphenicol, and increased killing of Caenorhabditis elegans compared to the inoculating strain (termed the P1 phenotype). The aims of this study were to characterize the differences between the P. aeruginosa P1 and P2 phenotypes in quorum sensing and competitiveness. We then determined the presence of the P2 phenotype among PAO1 strains from various laboratories. Results demonstrated that P2 cells display accelerated growth during early exponential phase and early activation of quorum-sensing systems and overcome the growth of P1 cells in a mixed population. Among eight PAO1 strains obtained from different laboratories, four exhibited the P2 phenotype. Of 27 mutants analyzed from the P. aeruginosa MPAO1 transposon library, 25 displayed P2 phenotypes. The P2 phenotype in both cases correlated with a lack of expression of mexE or mexF due to mutations in mexT and mexF genes. In summary, strains possessing the P2 phenotype are distributed among PAO1 strains commonly used across a variety of research laboratories. Genetically, they are characterized by various mutations in mexT or mexF.

Functional activity of allatotropin and allatostatin in the pupal stage of a holometablous insect, Tribolium castaneum (Coleoptera, Tenebrionidae)

Allatotropin (AT) and allatostatin (AS) neuropeptides are known to regulate the biosynthesis of juvenile hormones (JH) in insects. Furthermore, they possess myoregulatory and other activities in a wide range of insect species. The genome of Tribolium castaneum encodes two AS and one AT precursors. Here we cloned the cDNAs of the precursors, followed their expression patterns during the pupal stage, and established their putative roles in adult development and oviposition of the females using RNA interference (RNAi). Cloning of the cDNA and gene structure analyses of the Tc-AT gene confirmed that the gene is expressed in three mRNA isoforms. Real-time PCR data demonstrate that the Tc-AT isoforms and the AS genes, Tc-AS C and Tc-AS B, are expressed in discerning developmental and tissue-specific patterns. Single injections of dsRNAi (targeted against the Tc-AT, Tc-AS C, and Tc-AS B, respectively), into young pupae resulted in abnormal adult phenotypes, whereby about half of the animals (P1 phenotype) looked relatively normal, but the females laid low numbers of eggs. The other halves (P2) exhibited strong developmental defects with abnormal duration of the pupal stage, abnormal head and body sizes, short elytra, and incomplete sclerotization. Moreover, these females deposited no eggs and died within one week after emergence. Individual silencing of the Tc-AT mRNA isoforms showed that Tc-AT3 had the most disruptive influence on adult development and fecundity of the females. Our findings clearly indicate a significant role of AT and AS neuropeptides in the pupa. The distinct mechanisms of action, however, remain to be determined.

P1/P2 genotyping of known and novel null alleles in the P1PK and GLOB histo‐blood group systems

The rare but clinically important null phenotypes of the P1PK and GLOB blood group systems are due to alterations in A4GALT and B3GALNT1, respectively. A recently identified single-nucleotide polymorphism in Exon 2a of A4GALT predicts the common P1 and P2 phenotypes but rare variants have not been tested. The aim of this study was to analyze 84 p, P1 (k) , and P2 (k) samples, with special emphasis on unknown alleles and the P(1) /P(2) marker. Of these, 27 samples came from individuals not previously investigated genetically and were therefore subjected to sequencing of A4GALT or B3GALNT1, and a subset was tested by flow cytometry. The P(1) /P(2) genotyping linked 20 p-inducing mutations in A4GALT to P(1) or P(2) allelic background. Eight p alleles remain unlinked due to compound heterozygosity. For 23 of 25 P(k) samples, concordant results were observed: P1 (k) samples had at least one P(1) allele while P2 (k) had P(2) only. The two remaining samples typed as P1+ and P1+(w) but were genetically P(2) /P(2) . A tendency toward higher P(k) antigen expression was observed on P1 (k) cells compared to P2 (k) . In total, six previously unknown null mutations were found and characterized in A4GALT while four new changes were revealed in B3GALNT1. For the first time, p alleles were shown to occur on both P(1) and P(2) allelic backgrounds. Furthermore, P(1) /P(2) genotyping predicted the P1 (k) versus P2 (k) phenotype in more than 90% of globoside-deficient samples. The number of GLOB-null alleles was increased by 50% and several P1PK-null alleles were identified.

A rare p phenotype caused by a 26-bp deletion in α 1,4-galactosyltransferase gene

To delineate serological features and genetic basis for a rare p phenotype of P1Pk blood group system found in a Chinese individual. Serological assaying was carried out for a proband with unexpected antibody found in his serum using specific antibodies and panel cells. Coding regions and flanking introns of α 1,4-galactosyltransferase gene (A4GALT) associated with the p phenotype were screened with polymerase chain reaction and DNA sequencing. A rare p phenotype of the P1Pk blood group system has been identified with red blood cells from the proband, whose serum contained anti-Tja antibody which can agglutinate and hemolyze with other common red blood cells. Other members of the proband's family were all normal with P1 or P2 phenotype. DNA sequencing has identified in the proband a homozygous 26 bp deletion at position 972 to 997 of the A4GALT gene. The deletion has caused a shift of the reading frame, resulting in a variant polypeptide chain with additional 83 amino acid residues compared with the wild-type protein. Other family members were either heterozygous for above deletion or non-deleted. A 26 bp deletion at position 972 to 997 of the A4GALT gene has been identified in a Chinese individual with p phenotype.

Serological and genetic study of a pedigree featuring a rare p phenotype

To explore genetic background of a pedigree with a rare p phenotype from Guangdong province. The rare p phenotype was identified by a conventional serologic method. With genomic DNA of proband and family members extracted, exon 3 of alpha-(1,4)galactosyltransferase (A4GALT) gene was amplified with PCR and analyzed by direct sequencing. The mutation found in the pedigree was screened in a normal population using direct sequencing. The proband and 4 family members with the rare p phenotype have all carried a point mutation c.100G>A (p.Val34Ile) in combination with a deletion-insertional mutation c.418_428del11ins34(p.Gln139Trpfs*72), which renders a compound mutation of A4GALT gene. One family member with P2 phenotype has carried a same heterozygous mutation. Of the 100 healthy donors, 5 have carried a heterozygous point mutation c.100G>A, and none carried the deletion-insertional mutation c.418_428del11ins34. The rare p phenotype of the pedigree has resulted from a compound mutation of the A4GALT gene, which is in keeping with a recessive inheritance pattern of the p phenotype.

Intestinal Tissues Induce an SNP Mutation in Pseudomonas aeruginosa That Enhances Its Virulence: Possible Role in Anastomotic Leak

The most feared complication following intestinal resection is anastomotic leakage. In high risk areas (esophagus/rectum) where neoadjuvant chemoradiation is used, the incidence of anastomotic leaks remains unacceptably high (∼10%) even when performed by specialist surgeons in high volume centers. The aims of this study were to test the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become in vivo transformed to express a tissue destroying phenotype. We developed a novel model of anastomotic leak in which rats were exposed to pre-operative radiation as in cancer surgery, underwent distal colon resection and then were intestinally inoculated with Pseudomonas aeruginosa, a common colonizer of the radiated intestine. Results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (>60%; p<0.01) when colonized by P. aeruginosa compared to radiated tissues alone (0%). Phenotype analysis comparing the original inoculating strain (MPAO1- termed P1) and the strain retrieved from leaking anastomotic tissues (termed P2) demonstrated that P2 was altered in pyocyanin production and displayed enhanced collagenase activity, high swarming motility, and a destructive phenotype against cultured intestinal epithelial cells (i.e. apoptosis, barrier function, cytolysis). Comparative genotype analysis between P1 and P2 revealed a single nucleotide polymorphism (SNP) mutation in the mexT gene that led to a stop codon resulting in a non-functional truncated protein. Replacement of the mutated mexT gene in P2 with mexT from the original parental strain P1 led to reversion of P2 to the P1 phenotype. No spontaneous transformation was detected during 20 passages in TSB media. Use of a novel virulence suppressing compound PEG/Pi prevented P. aeruginosa transformation to the tissue destructive phenotype and prevented anastomotic leak in rats. This work demonstrates that in vivo transformation of microbial pathogens to a tissue destroying phenotype may have important implications in the pathogenesis of anastomotic leak.

A new look at an old case: An auto-anti-P with pseudo-LKE activity

Aims: LKE is a high-incidence, minor RBC glycosphingolipid, related to both Pk and P antigens. Approximately 1% individuals are LKE-negative. However, antibodies against LKE are rare, with only six cases mentioned in literature. Past examples of anti-LKE have relied on serologic testing, with no direct testing against RBC glycosphingolipid (GSL). To test a historical 'anti-LKE' against a panel of RBC and glycosphingolipid standards by high performance thin layer chromatography and standard serology. Methods: Serum samples included human polyclonal anti-LKE, alloanti-P, alloanti-PP1Pk and untransfused controls. Hemagglutination was performed by gel method with ficin-treated RBC of known LKE, P and P1 phenotype. P antigen expression was determined by titration with a well characterized alloanti-P. Antibody specificity was determined by incubating serum against glycosphingolipids on high performance thin layer chromatography plates. Results: The patient's serum reacted with most LKE+ RBC but not ficin-treated p, Pk, or LKE-negative donors, consistent with an anti-LKE. However, on direct testing, the patient's antibody failed to recognize monosialogalactosylgloboside, the LKE antigen. The patient's serum did recognize globoside (P) antigen. This was confirmed by hemagglutination, which showed a correlation between LKE phenotype, P antigen expression and serum reactivity. The patient's weak auto-anti-P was not inhibited by solubilized globoside. Conclusion: This historical anti-LKE is an auto-anti-P with 'pseudo-LKE' activity due to differences in P antigen expression between LKE+ and LKE-donors.

Developmentally early and late onset of Rps10 silencing in Arabidopsis thaliana: genetic and environmental regulation.

Transgene dosage, silencing competence of the transgene loci, and photoperiod conditions were found to regulate the onset and efficiency of Rps10 silencing in two independent transgenic lines of Arabidopsis thaliana. The Rps10 gene encodes the S10 protein which is part of the small subunit of mitochondrial ribosomes. Homozygous plants presented developmentally early onset of silencing, a very efficient decrease in the level of Rps10 transcripts, as well as a severe and uniform phenotype called P1. P1 plants either died during the vegetative growth phase or were rescued by reversion resulting from inactivation of silencing. A wide variety of morphological and developmental abnormalities observed within the hemizygous transformants allowed their classification into three categories P2, P3, and P4. The most severe and early was the P2 phenotype found in only one transgenic line and most probably resulting from high competence of the transgene loci. Developmentally late onset of silencing occurred only in the short day photoperiod and was characteristic for the P3 and P4 plants. This phenomenon was attributed to conditions favourable to silencing achieved in the short day photoperiod, e.g. a greatly prolonged vegetative phase accompanied by a gradual increase of the level of Rps10 transcripts. To the best of our knowledge, this is the first report indicating that the onset of silencing depends on the photoperiod conditions in A. thaliana.

Proteomic and mRNA Expression Chip Analysis of Acquired TRAIL-Resistance in Human HL60 Myeloid Leukemia Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a proapoptotic cytokine implicated in cancer cell surveillance. TRAIL induces apoptosis of target cells by the extrinsic, receptor-mediated, apoptotic pathway. A potential of TRAIL as cancer-specific therapeutic agent has been proposed, either as a single agent or in combination with chemotherapy agents. Development of TRAIL-resistant clones in the originally TRAIL-sensitive tumor cell population may be a critical complication of TRAIL based cancer therapy. Prolonged exposure of TRAIL-sensitive leukemia cell line, “wild-type” (WT) HL60 cells, to recombinant soluble TRAIL resulted in the establishment of resistant subclones. To get deeper insight into the molecular mechanism of acquired TRAIL resistance we compared expression profiles of TRAIL-sensitive HL-60 cells and TRAIL-resistant HL-60 subclones using microarray mRNA gene expression chips and 2D-PAGE followed by MS and MS/MS analysis. Using cluster analysis of microarray mRNA gene expression profiles of 6 individual HL60 resistant subclones we identified two molecular signatures of TRAIL resistance. When compared to WT HL60 TRAIL-sensitive mRNA expression the group of HL60 subclones designed as phenotype 1 (P1, n=3) had 1122 significantly upregulated and 985 significantly downregulated mRNAs and subclones designed as phenotype 2 (P2, n=3) had 1583 significantly upregulated and 1798 significantly downregulated mRNAs (p<0.05). The subclones of P1 and P2 phenotype shared 533 upregulated and 422 downregulated genes compared to HL60 WT cells. To identify differentially expressed proteins we performed 2D-PAGE of two representative subclones of P1 and P2 phenotypes (in five replicates) and compared them with HL60 WT cells. We detected and identified differentially expressed proteins involved in various aspects of cellular metabolism. Except for downregulation of chromobox protein homolog 5, a protein involved in the regulation of gene expression, and downregulation of Annexin A6 and protein disulfide-isomerase A3 precursor the TRAIL-resistant P1 and P2 subclones showed different protein expression profile. In P1 TRAIL-resistant cells we identified downregulation of proteins involved in energy metabolism, including pyruvate kinase, enolase, cytochrome c-reductase, NADH dehydrogenase and downregulation of proteins involved in proteasome activity, such as proteasome activator complex subunits 1 and 2. In P2 TRAIL-resistant cells we detected downregulation of important regulatory proteins, such as DNA replication licensing factor MCM7, proliferation-associated protein 2G4, replication protein A 32 kDa subunit. In both, P1 and P2, subclones we identified significant changes in cytoskeletal rearrangement proteins. In addition, several chaperones were differentially expressed, namely protein-disulfide isomerase, GrpE, HSC71 and HSP27, 78 kDa glucose-regulated protein precursor. Our data show two HL60 cell line-derived TRAIL resistant subclones, P1 and P2, have specific molecular signatures suggesting distinct mechanisms of TRAIL resistance. These distinct pathways for development of resistance to TRAIL-induced apoptosis are relevant for design of more effective strategies for leukemia therapy.