Sakae Tanosaki

Down-Regulation and Growth Inhibitory Role of C/EBPα in Breast Cancer

Purpose: CCAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that regulate proliferation and differentiation in a variety of tissues. The purpose of this study was to explore the possibility that C/EBPα is involved in breast cancer. Experimental Design: We quantified C/EBPα mRNA expression levels in 24 primary breast tumors, 16 normal breast samples, and 8 breast cancer cell lines using quantitative real-time reverse transcription-PCR assay. C/EBPα protein levels were further determined by immunohistochemical analysis. To examine the consequence of C/EPBα expression in breast cancer, we stably transfected an inducible C/EPBα expression vector into three breast cancer cell lines. Results: Low expression of C/EBPα mRNA was found in 83% of primary breast cancer samples. Immunohistochemical study further showed either a markedly reduced or undetectable expression of C/EBPα protein in 30% of breast cancer specimens. The other 70% of breast cancers had C/EBPα expression in both the cytoplasm and nucleus; in control, C/EBPα was localized to the nucleus in the normal ductal cells. C/EBPα expression was associated with estrogen- and progesterone receptor–negative status. Induction of C/EBPα expression in these cell lines resulted in growth inhibition accompanied by G0-G1 cell cycle arrest and reduced anchorage-independent cell growth. C/EBPα expression was associated with down-regulation of c-myc and up-regulation of p21, PPARγ, and the breast epithelial differentiation marker, maspin. Conclusions: These results suggest that reduced expression of C/EBPα may play a role in the development and/or progression of breast cancer.

Abnormality of c-kit oncoprotein in certain patients with chronic myelogenous leukemia--potential clinical significance.

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) chromosome and bcr/abl gene rearrangement which occurs in pluripotent hematopoietic progenitor cells expressing the c-kit receptor tyrosine kinase (KIT). To elucidate the biological properties of KIT in CML leukemogenesis, we performed analysis of alterations of the c-kit gene and functional analysis of altered KIT proteins. Gene alterations in the c-kit juxtamembrane domain of 80 CML cases were analyzed by reverse transcriptase and polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP). One case had an abnormality at codon 564 (AAT→AAG, Asn→Lys), and six cases had the same base abnormality at codon 541 (ATG→CTG, Met→Leu) in the juxtamembrane domain. Because the change from Met to Leu at codon 541 was a conservative one which was also observed in the normal population and normal tissues of CML patients, it probably represents a polymorphic variation. Although samples of hair roots and leukemic cells from the chronic phase of one CML patient showed no abnormality, an abnormality at codon 541 (ATG→CTG, Met→Leu) was found only at blastic crisis (BC) of this case. In the case with the abnormality at codon 564, the mutation was detected only in a sample of leukemic cells collected at BC. To examine the biological consequence and biological significance of these abnormalities, murine KITL540 and KITK563 expression vectors were introduced into interleukin-3 (IL-3)-dependent murine Ba/F3 cells to study their state of tyrosine phosphorylation and their growth rate. Ba/F3 cells expressing KITWT, KITL540 and KITK563 showed dose-dependent tyrosine phosphorylation after treatment with increasing concentrations of recombinant mouse stem cell factor (rmSCF). The cells expressing KITL540 and KITK563 were found to have greater tyrosine phosphorylation than cells expressing KITWT at 0.1 and 1.0 ng/ml of rmSCF. The Ba/F3 cells expressing KITK563 proliferated in response to 0.1 and 1.0 ng/ml of rmSCF as well as IL-3. The Ba/F3 cells expressing KITL540showed a relatively higher proliferative response to 0.1 ng/ml of rmSCF than the response of cells expressing KITWT. These mutations and in vitro functional analyses raise the possibility that the KIT abnormalities influence the white blood cell counts (P < 0.05) and survival (P < 0.04) of CML patients.

C|[sol]|EBP|[delta]| expression in a BCR-ABL-positive cell line induces growth arrest and myeloid differentiation

CCAAT/enhancer-binding proteins (C/EBPs) are a family of highly conserved transcription factors that have important roles in normal myelopoiesis as well as associated with myeloid disorders. The chronic myelogenous leukemia (CML) cell lines, KCL22 and K562, express exceptionally low levels of endogenous C/EBPs and provide a good model to test the effects of C/EBPs on myeloid differentiation. To explore the possibility that C/EBPδ can promote differentiation in BCR-ABL-positive cells, we generated stable KCL22 and K562 clones that expressed an inducible C/EBPδ gene. C/EBPδ expression resulted in G0/G1 proliferative arrest and a moderate increase in apoptosis of the KCL22 and the K562 cells. Within 4 days of inducing expression of C/EBPδ, myeloid differentiation of the CML blast cells occurred as shown by morphologic changes and induction of secondary granule-specific genes. We also showed that during granulocytic differentiation of KCL22 cells, the C/EBPδ protein was detected in immunocomplexes with both Rb and E2F1. Furthermore, expression of C/EBPδ was associated with downregulation of c-Myc and cyclin E and upregulation of the cyclin-dependent kinase inhibitor p27Kip1 in both the KCL22 and K562 cell lines. These results show that expression of C/EBPδ in BCR-ABL-positive leukemic cells in blast crisis is sufficient for neutrophil differentiation and point to the therapeutic potential of ectopic induction of C/EBPδ in the acute phase of CML.

Identifying progression-associated genes in adult T-cell leukemia/lymphoma by using oligonucleotide microarrays

Adult T‐cell leukemia/lymphoma (ATL) is associated with human T‐lymphotropic virus type‐1 (HTLV‐1). To understand the changes in expression that occur in the progression of chronic phase of ATL to acute crisis, the gene expression profiles of fresh ATL cells were compared in 4 pairs of samples (progression of chronic to acute phase in 3 patients, as well as 1 typical chronic phase sample vs. 1 typical acute phase sample) using high‐density oligonucleotide DNA arrays. We identified 203 genes that were commonly upregulated in acute vs. chronic phase samples including ribosomal proteins, proteosome subunits, eukaryotic translation factors, immunophilins, heat shock proteins and genes important for DNA replication. Additionally, we identified 91 commonly downregulated genes including immune molecules related to MHC and a phosphatase. Several of the genes were previously identified to be associated with the Tax protein of HTLV‐1. Some of the upregulated genes were located in amplified regions identified by comparative genomic hybridization in the corresponding chronic/acute ATL sample. Using real‐time quantitative PCR, we confirmed the array‐results in those specimens analyzed by microarray. These results demonstrated that distinct sets of genes that are known to be critical in cellular transformation and/or activation are up‐ or down‐regulated during the transition to the acute phase of ATL.

Effect of ligands of nuclear hormone receptors on sodium/iodide symporter expression and activity in breast cancer cells

Iodide uptake by normal and cancerous thyroid cells is an active process mediated by the sodium/iodide symporter (NIS). Using quantitative real-time RT-PCR, we found that all 22 fresh human breast cancer samples had very low NIS expression similar to levels in untreated MCF-7 breast cancer cells. 9-cis retinoic acid (9-cis RA), a ligand for both retinoic acid receptor (RAR)/retinoic X receptor (RXR) heterodimers as well as RXR/RXR homodimers, markedly induced NIS mRNA expression in MCF-7 breast cancer cells in a dose- and time-dependent fashion, with maximal levels occurring at 12 h. All-trans retinoic acid, ATRA, a RAR specific ligand had a similar potency. Among eight breast cancer cell lines, three out of four estrogen receptor (ER)-positive and zero of four ER-negative cell lines responded to 9-cis RA by increasing their expression of NIS. Combining a RAR with a RXR selective ligand enhanced both NIS mRNA expression and iodide uptake in MCF-7 cells. Similarly, a ligand for proliferator-activated receptor γ (PPARγ) when combined with 9-cis RA synergistically increased both NIS mRNA levels and iodide uptake in these MCF-7 cells. The iodide uptake was blocked by KClO4. In conclusions, these findings suggest that selected combinations of NHR ligands should be examined in a limited trial to determine if their administration to patients allows the use of radioactive iodine for diagnosis and possibly treatment of metastatic breast cancer.

Establishment of a cell line with variant BCR/ABL breakpoint expressing P180BCR/ABL from late-appearing Philadelphia-positive acute biphenotypic leukemia

In acute leukemia (AL) with a late‐appearing Philadelphia (la‐Ph) translocation, it is unclear whether these translocations arise from the same molecular event as classical Ph translocations. In order to elucidate the molecular events of la‐Ph and subsequent translocations of la‐Ph leukemia, we performed molecular analysis on the complex rearrangements, in a cell line, MY, which was established from bone marrow mononuclear cells of a patient with a la‐Ph acute biphenotypic leukemia. This la‐Ph, expressing an acute lymphoblastic leukemia (ALL)‐type BCR/ABL transcript, produces a novel P180BCR/ABL fusion protein reflecting deletion of 174 bases (58 amino acids) encoded by the a2 exon of the ABL gene. An immune complex kinase assay showed that this protein had autophosphorylation activity. Fluorescence in situ hybridization (FISH) in conjunction with G‐banding analysis revealed that the initial der(9)t(9;22)(q34;q11) progressed to a der(9)(9pter→9q34::22q11→22q13::5q11.2→5q15::10q23→10qter) by, first, a three‐way translocation among the der(9)t(9;22)(q34;q11), chromosome 5, and the normal chromosome 22, and then a subsequent translocation with chromosome 10. Moreover, both the end‐stage leukemic cells of the patient and the MY cell line had another translocation, t(X;12)(p11.2;p13). The 12p breakpoint was located near the ETV6 gene by analysis of pulsed‐field gel electrophoresis, but transcription of ETV6 was unaffected. Tumorigenicity analysis indicated that an additional translocation, t(2;3)(p16;q29), may have caused a more malignant clone, because only MY cells with the t(2;3)(p16;q29) were capable of growing subcutaneously in nude mice within 40 days. The molecular events of leukemogenesis and leukemic progression in the present la‐Ph AL occurred by accumulation of unique translocations. This cell line, MY, expressing a novel variant P180BCR/ABL protein with a deletion of the a2 exon of the ABL gene, may be useful for elucidating the pathophysiology of this fusion protein and for studying ETV6‐related leukemogenesis and t(2;3), as well as the molecular mechanisms of the complex translocations. Genes Chromosomes Cancer 23:227–238, 1998.

Heterogenous expression of bcr‐abl fusion mRNA in a patient with Philadelphia‐chromosome‐positive acute lymphoblastic leukaemia

We performed molecular and cytogenetic analysis on a 56‐year‐old woman with Philadelphia chromosome (Ph1)‐positive acute lymphoblastic leukaemia (ALL) having two types of major and minor bcr‐abl (M‐bcr‐abl, m‐bcr‐abl) fusion mRNA at diagnosis. In the course of her disease, unexpected heterogenous bcr‐abl fusion mRNA was detected by sequential analysis using the reverse transcription and polymerase chain reaction (RT‐PCR).

Possible transforming activity of interferon regulatory factor 2 in tumorigenicity assay of NIH3T3 cells transfected with DNA from chronic myelogenous leukemia patients

Little is known about the transforming gene identified in the genomic DNA of chronic myelogenous leukemia (CML) by the tumorigenicity assay. To detect a new transforming gene of CML, we re-investigated the transforming activity of interferon regulatory factor (IRF)-1 and -2 genes in the tumorigenicity assay of NIH3T3 cells transfected with genomic DNA of leukemic cells from 15 patients with CML (12 patients in the chronic phase, one in the blastic phase and two in both phases). We detected the functionally active IRF-2 gene only in the tumor DNA from two CML patients in the blastic phase. We did not detect integration of the IRF-1 gene in the DNA of any tumors derived from the CML patient samples, and also we detected no expression of human IRF-1 mRNA. Thus, NIH3T3 cells may have been transformed due to integration of the functionally active IRF-2 gene from CML patients in the blastic phase. We surmise that there is a possibility that the IRF-2 gene may be involved in the evolution of the blastic phase of CML.

Minimal Residual Disease in Acute Myelogenous Leukemia with PML/RARα or AML1/ETO mRNA and Phenotypic Analysis of Possible T and Natural Killer Cells in Bone Marrow

Here we studied minimal residual disease (MRD) of patients with acute myeloid leukemia (AML) who have PML/RARα or AML1/ETO as well as the phenotypic analysis of lymphocyte subsets involved in antitumor immunity. Eight patients in long-term (LT; 3 to 15 years) and 15 patients in short-term (ST; up to 3 years) remission were studied. Using the reverse transcription-polymerase chain reaction (RT) assay, the limit of detection was 10-5 to 10-6 for PML/RARα transcript and 10-4 to 10-5 for the AML1/ETO transcript. Simultaneously, T lymphocyte subsets and NK cells from the peripheral blood (PB) and bone marrow (BM) were investigated by flow cytometric analysis. Four of the eight patients in LT and 7 of the 15 patients in ST remission were MRD-positive. Although all MRD-positive patients in LT remission are still until now event-free, 3 of the 7 MRD-positive (MRD+) patients in ST remission soon relapsed. The total populations of CD4+, CD8+ and CD56+ [possible T-cell and natural killer (T/NK) populations] in the B...

Establishment of a cell line with AML1-MTG8, TP53, and TP73 abnormalities from acute myelogenous leukemia.

Gene alterations accumulate during the progression of acute myelogenous leukemia (AML) to a malignant clone. Here, a new myeloid cell line, designated YSK‐21, with the balanced t(8;21)(q22;q22) and the unbalanced der(1)t(1;17)(p36;q21), was established. YSK‐21 grows well in a medium containing recombinant human granulocyte colony‐stimulating factor (rhG‐CSF), granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF), or interleukin‐3 (rhIL‐3). Molecular analysis using the reverse transcriptase‐polymerase chain reaction (RT‐PCR) and fluorescence in situ hybridization (FISH) revealed that t(8;21)(q22;q22) resulted in an AML1‐MTG8 fusion transcript. FISH and spectral karyotyping (SKY) in conjunction with G‐banding analysis revealed a der(1)t(1;17)(p36;q21) chromosomal translocation, which appeared in the clone developed from the original leukemic cells. Molecular analysis of the TP73 gene on 1p36 and the TP53 gene revealed a deletion of one‐allele in TP73 with partial demethylation of another allele in the initial clone of YSK, and a point mutation consisting of an A→T substitution in codon 288 of the TP53 gene in the developed clone of YSK‐21. YSK‐21 cells, expressing aberrant AML1‐MTG8, TP53, and TP73 protein molecules, may be useful for elucidating the pathophysiology of these aberrant proteins and for studying the der(1)t(1;17)(p36;q21) chromosomal translocation.