Two populations of double minute chromosomes harbor distinct amplicons, the MYC locus at 8q24.2 and a 0.43-Mb region at 14q24.1, in the SW613-S human carcinoma cell line.

High-level amplifications observed in tumor cells are usually indicative of genes involved in oncogenesis. We report here a high resolution characterization of a new amplified region in the SW613-S carcinoma cell line. This cell line contains tumorigenic cells displaying high-level MYC amplification in the form of double minutes (DM(+) cells) and non tumorigenic cells exhibiting low-level MYC amplification in the form of homogeneously staining regions (DM(-) cells). Both cell types were studied at genomic and functional levels. The DM(+) cells display a second amplification, corresponding to the 14q24.1 region, in a distinct population of DMs. The 0.43-Mb amplified and overexpressed region contains the PLEK2, PIGH, ARG2, VTI1B, RDH11, and ZFYVE26 genes. Both amplicons were stably maintained upon in vitro and in vivo propagation. However, the 14q24.1 amplicon was not found in cells with high-level MYC amplification in the form of HSRs, either obtained after spontaneous integration of endogenous DM MYC copies or after transfection of DM(-) cells with a MYC gene expression vector. These HSR-bearing cells are highly tumorigenic. The 14q24.1 amplification may not play a role in malignancy per se but might contribute to maintaining the amplification in the form of DMs.

A set of proteins interacting with transcription factor Sp1 identified in a two-hybrid screening.

The two-hybrid system was used to isolate cDNA clones encoding polypeptides that interact with the N-terminal region (activation domains A, B and C) of the Sp1 transcription factor. Among the 65 collected clones, 43 contained cDNA fragments with open reading frames. They corresponded to 13 genes encoding proteins of known function and to 15 genes, the proteins of which have no known function. Six overlapping cDNA clones corresponded to the Hsc70 protein. Host cell factor (HCF-1) and the KIAA0461 gene (encoding a putative Zn-finger protein of unknown function) were both identified through the isolation of three overlapping cDNA clones. Two cDNA fragments encoding the same region of the SREBP-2 transcription factor were independently selected and two overlapping cDNA clones corresponded to the splicing factor SF3A120. Two different cDNA clones encoded the N- and C-terminal region of the Oct-1 transcription factor. Transcription factors Elf-1 and TIEG, as well as HSph2, the putative human homologue of a murine polyhomeotic gene, were each represented by a single clone. Noticeably, for the four identified transcription factors, the DNA-binding domain was excluded from the selected polypeptides. In vitro binding of the selected polypeptides to the Sp1 protein was demonstrated for the four transcription factors and for the SF3A120, Hsc70, HCF-1, HSph2 and pKIAA0461(245) proteins. Four other cDNA clones encoding polypeptides of unknown function were tested in the in vitro binding assay. All four polypeptides were found to interact with Sp1 in this assay.

Regulation of FGF-3 gene expression in tumorigenic and non-tumorigenic clones of a human colon carcinoma cell line.

The FGF-3 gene is constitutively expressed in tumorigenic clones from the SW613-S human colon carcinoma cell line but is silent in non-tumorigenic clones. We have investigated the transcriptional mechanisms responsible for this differential expression. Mapping of DNase I-hypersensitive sites throughout the FGF-3 gene and the region extending 15 kilobases upstream disclosed differences in the patterns obtained between tumorigenic and non-tumorigenic cells. Transient expression assays carried out with a reporter gene driven by FGF-3 promoter fragments of various lengths (0.143 to 11 kilobases) did not reproduce the differential regulation of the resident gene between the two cell types. The same constructs did exhibit a differential activity in stable transfectants, suggesting the involvement of a chromatin-based mechanism in this regulation. Under these conditions, even the 143-base pair minimal promoter fragment was able to drive the differential expression of the reporter gene. During the course of these analyses, several transcriptional modulatory elements (mainly activators) were identified in the FGF-3 upstream region and were found to colocalize with DNase I-hypersensitive sites. Moreover, a putative new promoter was discovered 6 kilobases upstream of FGF-3. Altogether, these data provide a basis for the elucidation of the complex regulation of the human FGF-3 gene.

Transcriptional mechanisms responsible for the overexpression of the keratin 18 gene in cells of a human colon carcinoma cell line.

The keratin 18 (K18) gene is overexpressed in cells of tumorigenic clones isolated from the SW613-S human colon carcinoma cell line, compared to cells of nontumorigenic clones. The isolated minimal promoter (TATA box and initiation site) of the K18 gene has by itself a differential activity in tumorigenic and nontumorigenic cells. An Sp1 binding site located upstream of the TATA box contributes to the high level of expression of the gene in tumorigenic cells. We report here that the Sp1 gene is not differentially expressed between the two cell types and that this is also the case for genes coding for factors of the preinitiation complex known to directly interact with the Sp1 protein. Further, DNase I footprinting experiments and mutagenesis analysis indicated that the mechanism responsible for the differential activity of the minimal K18 promoter apparently does not involve the binding of a factor to a specific sequence. During the course of these experiments, it was found that the initiation site of the K18 promoter is actually located 11 bp upstream of the +1 position previously reported and that the TATA box is the only essential element of the minimal promoter. Treatment of the cells with histone deacetylase inhibitors was more efficient at stimulating the activity of the K18 promoter in nontumorigenic cells than in tumorigenic cells. We propose that overexpression of the K18 gene in tumorigenic cells could result from of a high level of acetylation of histones and/or of factors controlling the activity of the transcription complex.

Apoptosis-prone phenotype of human colon carcinoma cells with a high level amplification of the c-myc gene.

Although apoptosis can be induced by the enforced expression of exogenously introduced c-myc genes, it is not clear whether overexpression resulting from the amplification of the resident c-myc gene in tumor cells is sufficient to induce apoptosis. We have investigated the relationship between c-myc gene amplification and the propensity of tumor cells to undergo apoptosis, using the SW613-12A1 and SW613-B3 cell lines, which are representatives, respectively, of tumorigenic and non-tumorigenic clones isolated from the SW613-S human colon carcinoma cell line. Tumorigenic clones are characterized by a high level of amplification and expression of the c-myc gene, whereas cells of non-tumorigenic clones have a small number of copies and a lower level of expression of this gene. Analysis of c-myc mRNA level in cells cultured under low serum conditions indicated that the expression of the gene is tightly regulated by serum growth factors in non-tumorigenic B3 cells, whereas it is poorly regulated in tumorigenic 12A1 cells, the level of mRNAs remaining relatively high in serum-starved 12A1 cells. Under these conditions, 12A1 cells showed clear evidence of apoptosis, whereas B3 cells were completely refractory to the induction of apoptosis. Moreover, the study of cell lines derived from non-tumorigenic apoptosis-resistant clones following the introduction by transfection of exogenous c-myc gene copies showed that they have acquired an apoptosisprone phenotype. Altogether, our results strongly suggest that deregulated c-myc expression due to high-level amplification confers an apoptosis-prone phenotype to tumor cells. The possible consequences of these observations for cancer therapy are discussed.

Deregulated expression of the keratin 18 gene in human colon carcinoma cells.

The keratin 18 (K18) gene is expressed at a normal level in cells of nontumorigenic clones derived from the SW613-S human colon carcinoma cell line, but is overexpressed in cells of tumorigenic clones. A high level of expression was also found in the cells from 10 of 15 other human colon carcinoma cell lines. The expression of the gene is downregulated in differentiating Caco-2 cells, resulting in a normal expression level. Determination of K18 mRNA half-life in growing and confluent Caco-2 cells indicated that this downregulation does not take place at a posttranscriptional level. The density of RNA polymerase molecules on the K18 gene, as measured in nuclear run-on experiments, is the same in growing and confluent Caco-2 cells, but the rate of synthesis of K18 transcripts in confluent Caco-2 cells, as determined by in vivo pulse-labeling, is 35% of that in growing cells. Nuclear run-on experiments carried out with nuclei prepared from growing or confluent Caco-2 cells treated with 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) indicated that a reduction in both the initiation and elongation rates of RNA polymerase molecules occurs on the K18 gene in confluent Caco-2 cells. This leads to a decreased rate of K18 transcript production with no reduction in the polymerase density on the gene. Evidence is provided that the mechanisms responsible for the differential expression of the K18 gene between tumorigenic and nontumorigenic SW613-S cells and between growing and differentiating Caco-2 cells share some similarities.

Sequence analysis of the transcription control region upstream of the human FGF-3 gene.

With the purpose of studying the transcriptional regulation of the human FGF-3 gene, we have cloned and determined the nucleotide sequence of the 11-kbp region flanking its 5' end. Analysis of the sequence disclosed the presence of multiple repetitive elements. Remarkably, all of them were found to have inserted in the same orientation as the FGF-3 gene, suggesting that the whole upstream region could play a role in the control of its transcription. Unique regions within the sequence were scanned for the presence of transcriptional regulatory elements. A potential "Initiator" sequence preceded by several motifs homologous to binding sites for transcription factors pinpointed a putative promoter, 6 kbp upstream of the ATG codon for the FGF-3 protein. A 250-nt sequence stretch surrounding the "Initiator" was found to display punctate homology with the first (P1) of the three promoters (P1, P2 and P3) of the mouse Fgf-3/int-2 gene, specifically in the region of the transcriptional start sites. These data should be useful in studying the mechanisms of regulation of the FGF-3 transcription unit.

Transplantation for Fanconi's anaemia: long-term follow-up of fifty patients transplanted from a sibling donor after low-dose cyclophosphamide and thoraco-abdominal irradiation for conditioning.

We describe the long-term follow-up of 50 Fanconi's anaemia patients who were transplanted from a related donor with a median follow-up of >6 years. The survival estimate was 74.4% at 54 months and 58.5% at 100 months. All patients were conditioned with low-dose cyclophosphamide and thoraco-abdominal irradiation. Acute graft-versus-host disease (GvHD) of grade II or more developed in 26 patients and chronic GvHD developed in 30/43 (69.9%) patients. The survival of patients without chronic GvHD (n = 13) was 100%. In addition to chronic GvHD, 20 pre-transplant transfusions was shown to have an adverse impact on survival by multivariate analysis (relative risk = 7.08, P = 0.0003). Prospective follow-up of growth and endocrine function could be performed in 31 patients. Of 20 boys, six have already reached normal puberty within the expected time. Among the 11 girls, three were at the pubertal age at the time of analysis. Growth retardation was common, whereas late complications (e.g. peripheral hypothyroidism, cataract) were rare. However, the most important long-term complication was the occurrence of cancer in seven patients (8-year projected incidence 24%). Among the 32 survivors, 27 (84.5%) had a normal and four a moderately reduced performance status, and all achieved complete engraftment with donor cells. Therefore transplantation was able to cure these patients who remain at high risk for developing late complications. Clearly, a genetic predisposition and chronic GvHD could have led to the development of these cancers. However, we cannot completely rule out irradiation as a cofactor in the genesis of these cancers, and therefore no longer use irradiation for the conditioning of Fanconi's anaemia patients.

Influence of CD34 cell selection on the incidence of mixed chimaerism and minimal residual disease after allogeneic unrelated donor transplantation.

Marrow transplantation from unrelated donors has been linked with an increased risk of graft-versus-host disease (GVHD). In an attempt to lower the risk of acute GVHD we used CD34 marrow cell selection for T cell depletion. Since T cell depletion has been linked to an increased risk of relapse and an increased risk of marrow failure, we used PCR amplification of minisatellite sequences to investigate donor cell engraftment and RT-PCR amplification of recurrent chromosomal translocations to investigate the residual disease post-transplant. Twenty-three patients who underwent BMT after positive selection of the CD34-positive cell population were studied. Results were then compared with those of 37 patients who underwent transplantation with unmanipulated marrow graft. Among the 23 patients who received CD34+ selected cell grafts, seven (30%) had evidence of full donor engraftment, 14 had evidence of residual recipient cells (61%), one had a non-take, and one autologous bone marrow recovery. Analysis of the chimaerism status post-transplant in 36 patients who received unmanipulated marrow grafts showed that 31 patients (86%) had evidence of full donor engraftment. The difference in the incidence of mixed chimaerism profile between patients who received unmanipulated marrow graft and those receiving CD34+ selected cell grafts was statistically significant (P< 0.01). Nine patients who received CD34+ selected cell grafts could be analysed for the presence of minimal residual disease post-transplant (one with t(9;22) acute lymphoblastic leukaemia and eight with CML). In the patient transplanted for a Ph-positive acute leukaemia, and in two out of the eight patients with CML, the search fora fusion transcript was consistently negative after transplantation. Among the six patients with evidence of residual disease, three patients also had a mixed chimaerism profile and were given donor lymphocyte infusions. Minimal residual disease study was performed post-transplant in 16 patients who received unmanipulated marrow grafts. In 10 of 14 patients with CML, and in two patients with acute leukaemia the search for a fusion transcript was consistently negative after transplantation. The difference in the incidence of minimal residual disease between patients who received an unmanipulated marrow graft and those receiving CD34+ selected cell grafts was not statistically significantly significant, but numbers of patients included in this analysis are still few. In conclusion, our study highlights the strong influence of graft manipulation on the incidence of mixed chimaerism after transplantation from an unrelated donor.

Hodgkin's disease of donor origin after allogeneic bone marrow transplantation for myelogeneous chronic leukemia.

Secondary malignancies (lymphomas, leukemias, and solid tumors) occurring after bone marrow transplantation are now more frequently reported, as the patients surviving the early phase of the graft and remaining free of their original disease are more numerous. Besides early Epstein-Barr virus-associated B-cell lymphoproliferative diseases, which are the most common type and most often of donor origin, few late-occurring lymphomas have been described that might represent a distinct entity. We report here a case of Hodgkin's disease developing 8 years after allogeneic bone marrow transplantation for chronic myelogeneous leukemia. Only two Hodgkin's diseases after allogeneic bone marrow transplantation have been reported in the literature so far. The case we report is of interest because of its donor origin and its association with Epstein-Barr virus infection.

Identification of enhancer-like elements in the ribosomal intergenic spacer of Odontophrynus americanus 2n and 4n (Amphibia, Anura).

Repetitive elements are found in the ribosomal intergenic spacer (IGS) of most organisms. A particularly complex pattern of internal repetition occurs in the IGSs of O. americanus 2n and 4n, which are composed of several types of BamHI subrepeats (B-SRs). The most repetitive one is approximately 87 bp long, and is highly represented in the IGS variants of these amphibians. Sequence analyses of six diploid and two tetraploid B-SRs show 87% and 86% homology, respectively, and related secondary structure predictions. The comparison of the 2n and 4n B-SR sequences aligned with the 81 bp enhancer of Xenopus laevis reveals 36% homology. Furthermore, other B-SR features like size, number, and secondary structures resemble those of Xenopus enhancers, suggesting that B-SRs may function as regulators of O. americanus rDNA transcription. The present data also corroborate the close evolutionary relationship between 2n and 4n O. americanus species.

Detection of maternal cells in human fetal blood during the third trimester of pregnancy using allele-specific PCR amplification.

Using a highly sensitive allele-specific PCR amplification method, we have previously shown that maternal cells could be detected in all 10 cord bloods tested. This raised the question of whether maternal cells are released into cord blood during the process of delivery or whether they are already present during pregnancy. We have now used the same PCR method to detect the presence of maternal cells in nine fetal blood samples collected at different gestational ages. Maternal cells were detected in eight samples obtained between 24 and 35 weeks of gestation. They were estimated to amount between 10(-4) and 10(-5) of nucleated fetal blood cells. In two cases mononuclear and polymorphonuclear cell fractions were separated by Ficoll gradient centrifugation and maternal cells were detected as comparable levels in both fractions. Maternal cells could not be detected in the one fetal blood sample obtained at 20 weeks of gestation, suggesting that maternal cells could appear at detectable levels in fetal blood during the third trimester of pregnancy. These results are discussed in terms of materno-fetal immune tolerance and of transmission of viruses (and more specifically of the human immunodeficiency virus) from mother to child.

Analysis of the size distribution of first-strand cDNA molecules.

We have developed a method to analyze the size distribution of the first-strand cDNA molecules corresponding to given mRNA species. First-strand molecules synthesized from cytoplasmic polyadenylated RNAs are separated by electrophoresis on an alkaline agarose gel, and a Southern blot hybridization is performed. As an example, we analyzed the first-strand molecules corresponding to the human c-myc mRNAs. This method can be used to determine whether full-length, first-strand molecules corresponding to an mRNA species to be cloned are synthesized efficiently. Interestingly, this method allows one to analyze full-length, first-strand cDNA molecules with a much higher resolution than Northern blot analysis of mRNA molecules. This method can therefore be used to discriminate between the multiple mRNA species transcribed from a given gene or the homologous mRNA species transcribed from a given gene family.

Characterization of new human c-myc mRNA species produced by alternative splicing.

Characterization of two human c-myc cDNAs corresponding to the mRNAs 2.5 and 3.1 kb in length transcribed from P0 previously demonstrated the existence of alternative acceptor sites at the end of intron 1 and intron 2, respectively [Bentley, D.L. and Groudine, M. (1986) Mol. Cell. Biol. 6, 3481-3489]. We investigated the use of these alternative acceptor sites in each c-myc mRNA species. We characterized cDNAs corresponding to c-myc mRNAs transcribed in the SW613-S human carcinoma cell line. The use of the alternative acceptor site at the end of intron 1 was demonstrated in two out of 10 cDNAs corresponding to the 3.1-kb mRNA transcribed from P0 and in three out of 10 cDNAs corresponding to the mRNAs transcribed from P1 or P2. The use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from P0. The mRNAs resulting from the use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from P0. The mRNAs resulting from the use of this acceptor site would encode for a variant form of the p67 polypeptide lacking one amino-acid residue. Conversely, the use of the alternative acceptor site at the end of intron 2 was not found in any of the cDNAs corresponding to the mRNAs transcribed from P0 (0/10), from P1 or P2 (0/10) and from P3 (0/10). In the course of this study, we isolated a cDNA corresponding to another new c-myc mRNA species. This mRNA is produced by alternative splicing within intron 1 and encodes only for p64.

A highly sensitive polymerase chain reaction method reveals the ubiquitous presence of maternal cells in human umbilical cord blood.

Umbilical cord blood is considered an alternate source of hematopoietic stem cells in bone marrow transplantation. However, its use might be hampered by contamination of neonatal blood with maternal cells, which could contribute unacceptably to graft-vs.-host disease (GVHD) after transplant. In a previous study (Socié et al., Blood 83:340, 1994), we used polymerase chain reaction (PCR) amplification of minisatellite sequences (sensitivity 1-0.1%) to address the question of this contamination. In a single case among 47 analyzed, we were able to detect a maternal-specific allele in the cord blood sample. We have now studied the same cord samples using a highly sensitive, allele-specific PCR amplification method. A maternal allele could be discriminated from neonate alleles in 10 cases and maternal cells were detected in all 10 cord blood samples. These cells amounted to 10(-4) to 10(-5) of cord blood nucleated cells. In three cases, cord blood separated cell subpopulations could be analyzed and were found to contain maternal cells at about the same level. The presence of maternal cells at such a low level in cord blood samples probably would have no effect on GVHD in a clinical setting of transplantation but raises interesting questions in terms of materno-fetal immune tolerance and transmission of viruses (in particular human immunodeficiency virus) from infected mother to child.

Cloning and sequencing of a tpr-met oncogene cDNA isolated from MNNG-transformed human XP fibroblasts.

A rearranged tpr-met oncogene was identified in a MNNG-transformed human Xeroderma pigmentosum (XP) cell line (ASKMN). A 2016 bp cDNA was cloned and sequenced, disclosing an ORF with a coding capacity for a 523 aa protein. The sequence of this tpr-met cDNA was very similar to that previously reported in another human MNNG-transformed cell line (MNNG-HOS).

Studies on hemopoietic chimerism following allogeneic bone marrow transplantation in the molecular biology era.

Donor hematopoiesis or donor chimerism in the host following allogeneic bone marrow transplantation (BMT) has appeared crucial to the engraftment process. However, as molecular techniques exploiting neutral variation in human genetic material have been used in the study of chimerism, the detection of residual host cells or mixed hemopoietic chimerism has indicated that donor chimerism is not obligatory following BMT. This review focuses on the detection and significance of mixed chimerism (MC) in patients transplanted for both malignant and non-malignant hemopoietic disease and attempts to tease out the contribution of MC to engraftment, leukemia relapse, graft rejection and long-term disease-free survival.