Micheline Giphart-Gassler

Analysis of Gene Expression Using Gene Sets Discriminates Cancer Patients with and without Late Radiation Toxicity

Background Radiation is an effective anti-cancer therapy but leads to severe late radiation toxicity in 5%–10% of patients. Assuming that genetic susceptibility impacts this risk, we hypothesized that the cellular response of normal tissue to X-rays could discriminate patients with and without late radiation toxicity. Methods and Findings Prostate carcinoma patients without evidence of cancer 2 y after curative radiotherapy were recruited in the study. Blood samples of 21 patients with severe late complications from radiation and 17 patients without symptoms were collected. Stimulated peripheral lymphocytes were mock-irradiated or irradiated with 2-Gy X-rays. The 24-h radiation response was analyzed by gene expression profiling and used for classification. Classification was performed either on the expression of separate genes or, to augment the classification power, on gene sets consisting of genes grouped together based on function or cellular colocalization. X-ray irradiation altered the expression of radio-responsive genes in both groups. This response was variable across individuals, and the expression of the most significant radio-responsive genes was unlinked to radiation toxicity. The classifier based on the radiation response of separate genes correctly classified 63% of the patients. The classifier based on affected gene sets improved correct classification to 86%, although on the individual level only 21/38 (55%) patients were classified with high certainty. The majority of the discriminative genes and gene sets belonged to the ubiquitin, apoptosis, and stress signaling networks. The apoptotic response appeared more pronounced in patients that did not develop toxicity. In an independent set of 12 patients, the toxicity status of eight was predicted correctly by the gene set classifier. Conclusions Gene expression profiling succeeded to some extent in discriminating groups of patients with and without severe late radiotherapy toxicity. Moreover, the discriminative power was enhanced by assessment of functionally or structurally related gene sets. While prediction of individual response requires improvement, this study is a step forward in predicting susceptibility to late radiation toxicity.

Identification of RUNX1/AML1 as a classical tumor suppressor gene

Based on our previous results indicating the presence of a tumor suppressor gene (TSG), chromosome 21 was analysed for loss of heterozygosity (LOH) in 18 patients with acute myeloid leukemia (17, AML-M0; one, AML-M1). Allelotyping at polymorphic loci was performed on purified material, allowing unequivocal detection of allelic loss and homozygous deletions. Six AML-M0 patients shared a common region of LOH harboring a single gene: RUNX1 (AML1), the most frequent site of translocations in acute leukemia and a well-known fusion oncogene. Fluorescence in situ hybridization allowed the identification of deletions with breakpoints within RUNX1 in two patients as the cause of LOH. In the four others the LOH pattern and the presence of two karyotypically normal chromosomes 21 were in line with mitotic recombination. Further molecular and cytogenetic analyses showed that this caused homozygosity of primary RUNX1 mutations: two point mutations, a partial deletion and, most significantly, a complete deletion of RUNX1. These findings identify RUNX1 as a classical TSG: both alleles are mutated or absent in cancer cells from four of the 17 AML-M0 patients examined. In contrast to AML-M0, the AML-M1 patient was trisomic for chromosome 21 and has two mutated and one normal RUNX1 allele, suggesting that the order of mutagenic events leading to leukemia may influence the predominant tumor type.

Hemizygous deletions in the HLA region account for loss of heterozygosity in the majority of diffuse large B-cell lymphomas of the testis and the central nervous system.

Loss of heterozygosity (LOH) is a major mechanism for inactivation of tumor‐suppressor genes and has been observed in various solid tumors and lymphomas. The human leukocyte antigen (HLA) region is located at chromosome band 6p21.3, and loss or alteration of this region may provide tumor cells with a mechanism to escape from the immune system. We previously identified small homozygous deletions within the HLA class II region in many of the diffuse large B‐cell lymphomas (DLCLs) of the central nervous system (CNS) and the testis. In the present study, we focused on the mechanism leading to LOH in the HLA region. Twenty microsatellite markers, of which 12 were specific for HLA, were applied on 11 extranodal DLCLs of the CNS and 28 of the testis. Additionally, fluorescence in situ hybridization with seven HLA‐specific probes and a centromere 6–specific probe was performed on 20 cases to study the mechanism of LOH. In contrast to previously published data on spontaneously mutated lymphoblastoid cell lines, intrachromosomal hemizygous deletion, not mitotic recombination, was the major cause of LOH of the HLA region in these lymphomas. However, opposed to data in colorectal cancer, these deletions were rarely (one of nine cases) associated with an interchromosomal rearrangement such as a translocation.

Chromosome loss with concomitant duplication and recombination both contribute most to loss of heterozygosity in vitro

Loss of heterozygosity (LOH) plays an important role in the expression of recessive mutations in mammalian cells. To gain insight into the rate and mechanisms of LOH the autosomal HLA‐A gene was used as a model system. Spontaneous HLA‐A2 mutants originated with a rate of respectively 4.1 × 10−6 and 6.9 × 10−6 per cell per generation in TK6 and WI‐L2‐NS, two isogenic lymphoblastoid cell lines which differ in TP53 status. The rate of loss of HLA‐A2 is 10–50 times higher compared to the mutation rate of the X‐linked HPRT gene. The homozygous TP53 mutation in WI‐L2‐NS had no effect on the rate of HLA‐A2 loss or the spectrum of these mutations. Microsatellite analysis of most of the HLA‐A2 mutants (84%) showed LOH for multiple markers on chromosome arm 6p telomeric of a recombination breakpoint, LOH for all 6p markers, or LOH for markers on both the 6p‐ and 6q‐arms. Cytogenetic analysis showed that these mechanisms gave mutant cells which harbored two intact chromosomes 6 and which were indistinguishable from non‐mutant cells. Therefore, loss of HLA‐A2 is mainly caused by somatic recombination (33–50%) or chromosome loss with duplication of the remaining chromosome (34–40%). These findings correspond to the mechanisms behind loss of the wild‐type RB1 allele in retinoblastoma and suggest that both somatic recombination and chromosome loss followed by duplication contribute to tumorigenesis. Genes Chromosomes Cancer 21:30–38, 1998.

Thermo-inducible expression of cloned early genes of bacteriophage Mu

An EcoRI fragment, containing approx. 5100 base pairs (bp) of the immunity-end of bacteriophage Mu, was inserted into the multicopy plasmid pMB9 by in vitro recombination. The expression of early Mu genes, located on the cloned fragment, is thermo-inducible because of the presence of the ts mutation in gene c. The isolation of a transformant harbouring the recombinant plasmid, pGP1, was possible only when expression of Mu genes was prevented. pGP1 can be maintained at 28 degrees C at high copy number, but at 42 degrees C the pGP1 containing cells are killed due to the expression of the kil gene of Mu. The following Mu genes are present on pGP1: the ner gene, the integration and replication genes A and B, the cim gene, and the kil gene. pGP1 containing cells do not show Gam and Sot activity at 42 degrees C, therefore the leftmost EcoRI site on the Mu DNA is located between genes kil and gam or sot, or within the gam or sot gene.

Gene expression profiling of minimally differentiated acute myeloid leukemia: M0 is a distinct entity subdivided by RUNX1 mutation status

Minimally differentiated acute myeloid leukemia (AML-M0) is defined by immature morphology and expression of early hematologic markers. By gene expression profiling (GEP) and subsequent unsupervised analysis of 35 AML-M0 samples and 253 previously reported AML cases, we demonstrate that AML-M0 cases express a unique signature that is largely separated from other molecular subtypes. Hematologic transcription regulators such as CEBPA, CEBPD, and ETV6, and the differentiation associated gene MPO appeared strongly down-regulated, in line with the primitive state of this leukemia. AML-M0 frequently carries loss-of-function RUNX1 mutation. Unsupervised analyses revealed a subdivision between AML-M0 cases with and without RUNX1 mutations. RUNX1 mutant AML-M0 samples showed a distinct up-regulation of B cell-related genes such as members of the B-cell receptor complex, transcription regulators RUNX3, ETS2, IRF8, or PRDM1, and major histocompatibility complex class II genes. Importantly, prediction with high accuracy of the AML-M0 subtype and prediction of patients carrying RUNX1 mutation within this subtype were possible based on the expression level of only a few transcripts. We propose that RUNX1 mutations in this AML subgroup cause lineage infidelity, leading to aberrant coexpression of myeloid and B-lymphoid genes. Furthermore, our results imply that AML-M0, although originally determined by morphology, constitutes a leukemia subgroup.

Structural polypeptides and products of late genes of bacteriophage Mu: Characterization and functional aspects☆

Abstract This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion. In vitro reconstitution of infectious phage particles has shown that four genes ( E , D , I , J ) control the formation of phage heads and that a cluster of eight genes ( K , L , M , N , P , Q , R , S ) controls the formation of phage tails. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10 3 M r ); H (64 × 10 3 M r ); F (54 × 10 3 M r ); G (16 × 10 3 M r ); L (55 × 10 3 M r ); N (60 × 10 3 M r ); P (43 × 10 3 M r ) and S (56 × 10 3 M r ). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V , W or R (41.5 × 10 3 M r , and 45 × 10 3 M r ); U (20.5 × 10 3 M r ) and of genes located in the β region (24 × 10 3 M r (gp gin ) and 37 × 10 3 M r (possibly gp mom )). Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10 3 , a second head polypeptide of 54 × 10 3 (gp F ) and two major tail polypeptides with molecular weights of 55 × 10 3 and 12.5 × 10 3 (gp L and gp Y , respectively). In addition, there are five minor components of the tail (including gp N , gp S and gp U ) and approximately seven minor components of the head structure of the virion (including gp H ).

Polypeptides encoded by the early region of bacteriophage Mu synthesized in minicells of Escherichia coli

Abstract The polypeptides encoded by the early region of the Mu genome (comprising approximately 8000 base-pairs of DNA extending from the immunity encoding end of the linear Mu genome) have been determined. Minicells were infected by wildtype, suppressor-sensitive-mutation-carrying Mu phages and by λpMu hybrid phages. Comparison of the polypeptides synthesized in these infected minicells with polypeptides synthesized in minicells containing recombinant plasmids, which carry the first 5100 base-pairs of the early region of Mu DNA, have permitted a definitive correlation of the region of DNA with the encoded gene product. The Mu represser protein, gpc (Mr, 26,000), is encoded by approximately 900 base-pairs of DNA extending from the c-end of the genome † . The region 1300 to 4300 base-pairs encodes gpA (Mr, 70,000) and gpB (Mr, 33,000) and the region 4300 to 5100 basepairs encodes gpcim (Mr, 7000) and gpkil (Mr, 8000). A 14,000 Mr polypeptide, which is probably gpgam, is located in the region 4800 to 5400 base-pairs, and three polypeptides of molecular weights 20,500, 19,000 to 22,000 and 11,500 are located in the region 5200 to 6600 base-pairs. One of these polypeptides is gpsot. The basis for the apparent range of molecular weights (19,000 to 22,000) exhibited by one of the polypeptides encoded in this region is unknown. The region 6200 to 8600 base-pairs encodes three polypeptides (Mr = 10,000, 10,000 and 8000) of unknown function. The polypeptides identified as early polypeptides from their location on the Mu genome are also those polypeptides detected earliest after infection of minicells and are the major translation products of Mu RNA synthesized in minicells infected in the presence of chloramphenicol. These early polypeptides, assuming no overlapping genes, require the expression of approximately 80% of the coding capacity of the early region of the Mu genome.

Lack of genetic and epigenetic changes in meningiomas without NF2 loss.

Approximately 60% of sporadic meningiomas are caused by inactivation of the NF2 tumour suppressor gene. The causative gene for the remaining meningiomas is unknown. Previous studies have shown that these tumours have no recurrent karyotypic abnormalities. They differ from their NF2‐related counterparts in that they are more often of the meningothelial subtype and are located preferentially in the anterior skull base. To gain more insight into the aetiology of these tumours, we studied genetic and epigenetic alterations in 25 meningiomas without NF2 involvement. We first established a genome‐wide allelotype using 3 microsatellite markers per chromosome arm. Loss of heterozygosity (LOH) was detected at a low frequency and no indication for the location of putative tumour suppressor genes could be established. We next screened the subtelomeric regions by using 2–3 polymorphic markers close to each telomere. Again no evidence for LOH of a particular chromosome arm was obtained, and no LOH was found in the genomic regions containing the NF2‐related ERM family members ezrin and radixin, DAL‐1, protein 4.1R, and TSLC1. Mutations in the X‐chromosome based family member, moesin, were analysed by SSCP and were not detected. Microsatellite instability was studied using 6 commonly used markers but none of these was altered in any meningioma. Methylation was detected in 5 of 16 genes (NF2, p14ARF, CDH1, BRCA1, RB1) previously shown to be silenced in a variety of tumour types. However, methylation percentages for these genes were generally higher in a group of NF2‐related meningiomas, with the exception of the BRCA1 gene. The NF2 gene was methylated in only 1 of 21 tumours. In conclusion, meningiomas with an intact NF2 gene have a normal karyotype and no obvious genetic or epigenetic aberrations, suggesting that the gene(s) involved in the pathogenesis of these tumours are altered by smaller events than can be detected with the techniques used in our study. Copyright

Isolation and molecular characterization of spontaneous mutants of lymphoblastoid cells with extended loss of heterozygosity.

The human major histocompatibility complex comprising the HLA class I and II genes provides a versatile source of natural heterozygous loci. This polymorphic genetic system allows analysis of the mechanistic aspects of loss of heterozygosity (LOH), a major phenomenon observed at tumor suppressor genes in human cancer cells. Four lymphoblastoid cell lines, ORI, TK6, WI-L2-NS and VH, were used to adjust current HLA immunoselection protocols to quantify loss of HLA-A2 in human lymphoblastoid cell lines. The modified selection protocol was used to isolate independent spontaneous HLA-A2 mutants from the lymphoblastoid cell line ORI. The frequency of spontaneous loss of HLA-A2 in ORI was 1.7 x 10(-5). By HLA typing 35 spontaneous HLA-A2 mutants, we showed that 74% of the HLA-A2 mutants also lost expression of the HLA-B allele, which is located on the same haplotype as HLA-A2. Microsatellites on both arms of chromosome 6 were used for molecular characterization of the spontaneous HLA-A2 mutants. Loss of heterozygosity at various loci on the p-arm or loss of an entire chromosome 6 was found in 80% of the mutants. Surprisingly, it appeared that a presumed mitotic recombination event in the cell line ORI itself had resulted in homozygosity of all markers distal from the HLA locus up to the telomere. This greatly limited the detection of mitotic recombination, resulting in LOH up to the telomere, on the short arm of chromosome 6 in this cell line. However, gene dosage analysis detected two copies of the remaining D6S265 allele in mutants which showed LOH at various loci along the p-arm. This suggested that recombination resulted in LOH in these mutants. The lymphoblastoid cell line TK6 did contain informative microsatellites along the complete chromosome 6. Mutants of TK6 either retained heterozygosity of all p-arm markers, showed LOH of all p-arm markers or showed loss from a breakpoint up to the telomere. These data indicate that recombination and chromosome loss both are important mechanisms involved in loss of the HLA-A2 allele in vitro. Such mechanisms may be involved in LOH in vivo and contribute to loss of tumor suppressor alleles.