Review of clinical, cytogenetic, and molecular aspects of Ph-negative CML

Abstract Between 1985 and 1989, many cases of Philadelphia (Ph) chromosome negative chronic myelogenous leukemia (CML) were reported. For this review, the following selection criteria were used: the original articles on Ph-negative cases should provide clinical, hematologic, cytogenetic as well as molecular data. In addition, eight unpublished cases of Ph-negative CML are included that were studied in our institute during the last two years. Our purpose was to correlate presence or absence of the Ph rearrangement with the clinical features in an attempt to test whether the entity “Ph-negative CML” really exists and to identify the pathologic characteristics, frequency of occurrence, prognosis for survival, and underlying molecular mechanisms. Data on Ph-negative CML patients were compared with data on Ph-positive CML, atypical CML (aCML), and chronic myelomonocytic leukemia (CMMoL), reported in the same papers as the Ph negative patients. Essential for comparison of data from the different investigators appeared to be a clear description of criteria they used to establish the diagnosis CML, or alternatively a complete presentation of data for all patients reported in the articles. In most cases. Ph-negative CML was distinguishable from CMMoL and aCML, using simple criteria, e.g., differential count of peripheral blood and absence of dysplasia in the bone marrow. Cytogenetic analysis showed normal karyotype in most cases of Ph-negative CML. Interestingly, in cases with abnormal karyotype, chromosome 9 band q34 was relatively frequently involved in translocations with other chromosomes than chromosome 22, suggesting a variant Ph translocation not visible by cytogenetic techniques. This assumption was confirmed by molecular analysis, demonstrating bcr-abl rearrangement in 9 out of 10 of the latter cases. Results of cytogenetic and molecular investigations in 136 cases of Ph-negative CML reviewed in this article clearly indicated that molecular techniques are valuable tools for identification of bcr-abl rearrangements, indicative for the Ph translocation. The different mechanisms responsible for bcr-abl rearrangement in Ph-negative CML patients are discussed. The question remains whether all Ph-negative CML patients will have bcr-abl rearrangements, or whether alternative mechanisms will be identified that are responsible for this disease

The restricted expression pattern of the POU factor Oct-6 during early development of the mouse nervous system

Oce-6 is a POU transcription factor that is thought to play a role in the differentiation of cells of neuroectodermal origin. To investigate whether the Oct-6 protein could play a role in the establishment of neuroectoderm in vivo we studied the expression of the Oct-6 protein during early mouse development. Expression is first observed in the primitive ectoderm of the egg cylinder stage embryo. In gastrulating embryos, Oct-6 protein is found in the extra-embryonic ectoderm of the chorion and the anterior ectoderm of the embryo proper. As development proceeds, Oct-6 expression becomes more restricted to the anterior medial part of the embryo until Oct-6 positive cells are observed only in the neural groove of the headfold stage embryo. In the late headfold stage embryo, Oct-6 expression is detected in the neuroepithelium of the entire brain and later is restricted to a more ventral and anterior position. As the anterior neuropore closes, Oct-6 protein is detected in a segment-like pattern in the mid- and forebrain. Thus, the expression pattern of the Oct-6 gene agrees with a role for the Oct-6 protein in the establishment and regional specification of the neuroectoderm in vivo. The two waves of widespread induction of the Oct-6 gene, one in the primitive ectoderm and another in the primitive brain, both followed by a progressive restriction in the expression patterns suggest a mechanism for the regulation of the gene

Translocation of BCR to chromosome 9: A new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leukemia

Leukemic cells from two patients with Philadelphia-negative chronic myeloid leukemia (CML) were investigated: I) Cytogenetics showed a normal 46.XY karyotype in both cases, 2) molecular studies revealed rearrangement of the M-BCR region and formation of BCR-ABL fusion mRNA with b2a2 (patient I) or b3a2 (patient 2) configuration, and 3) fluorescence in situ hybridization (FISH) demonstrated relocation of the 5′ BCR sequences from one chromosome 22 to one chromosome 9. The ABL probe hybridized to both chromosomes 9 at band q34, while two other probes which map centromeric and telomeric of BCR on 22q 11 hybridized solely with chromosome 22. For the first time, a BCR-ABL rearrangement is shown to take place on 9q34 instead of in the usual location on 22q 11. A rearrangement in the latter site is found in all Ph-positive CML and in almost all investigated CML with variant Ph or Ph-negative, BCR-positive cases. The few aberrant chromosomal localizations of BCR-ABL recombinant genes found previously were apparently the result of complex and successive changes. Furthermore in patient 2, both chromosomes 9 showed positive FISH signals with both ABL and BCR probes. Restriction fragment length polymorphism (RFLP) analysis indicated that mitotic recombination had occurred on the long arm of chromosome 9 and that the rearranged chromosome 9 was of paternal origin. The leukemic cells of this patient showed a duplication of the BCR-ABL gene, analogous to duplication of the Ph chromosome in classic CML. In addition they had lost the maternal alleles of the 9q34 chromosomal region. The lymphocytes of patient 2 carried the maternal chromosome 9 alleles and were Ph-negative as evidenced by RFLP and FISH analyses, respectively. © 1993 Wiley-Liss, Inc

The Leukemia-Associated Fusion Protein MN1-TEL Blocks TEL-Specific Recognition Sequences

The leukemia-associated fusion protein MN1-TEL combines the transcription-activating domains of MN1 with the DNA-binding domain of the transcriptional repressor TEL. Quantitative photobleaching experiments revealed that ~20% of GFP-tagged MN1 and TEL is transiently immobilised, likely due to indirect or direct DNA binding, since transcription inhibition abolished immobilisation. Interestingly, ~50% of the MN1-TEL fusion protein was immobile with much longer binding times than unfused MN1 and TEL. MN1-TEL immobilisation was not observed when the TEL DNA-binding domain was disrupted, suggesting that MN1-TEL stably occupies TEL recognition sequences, preventing binding of factors required for proper transcription regulation, which may contribute to leukemogenesis

Cre-mediated site-specific translocation between nonhomologous mouse chromosomes

Chromosome rearrangements, such as large deletions, inversions, or translocations, mediate migration of large DNA segments within or between chromosomes, which can have major effects on cellular genetic control. A method for chromosome manipulation would be very useful for studying the consequences of large-scale DNA rearrangements in mammalian cells or animals. With the use of the Cre-loxP recombination system of bacteriophage P1, we induced a site-specific translocation between the Dek gene on chromosome 13 and the Can gene on chromosome 2 in mouse embryonic stem cells. The estimated frequency of Cre-mediated translocation between the nonhomologous mouse chromosomes is approximately 1 in 1200-2400 embryonic stem cells expressing Cre recombinase. These results demonstrate the feasibility of site-specific recombination systems for chromosome manipulation in mammalian cells in vivo, breaking ground for chromosome engineering

Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.

The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of beta-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of