Establishment And Characterization Of Two Human Acute Myelomonocutic Leukemia Cell Lines, JIH-3 And JIH-6 With Del(7p) And T(9;22), Respectively

ObjectiveLeukemia cell lines sustained proliferation in vitro with preservation of most cellular features during long-term culture. They represent reliable model systems and provide powerful tools for the investigation of leukemogenesis, the analysis of drug effect and the research of biotechnology. To date, more than 1000 leukemia-lymphoma (LL) cell lines have been reported. The majority of them belong to lymphoid-originated cell lines, while myelomonocytic leukemias only account for a minor percentage. It is regretted that large panels of LL line cells are not available because they are not sufficiently characterized and are incompletely described in the literature. We establish two novel human myelomonocytic leukemia cell lines with specific chromosomal abnormality by culturing the primary leukemia cells from a acute myeloid leukemia(AML)-M4 and a chronic myeloid leukemia (CML) in myeloid blast crisis patients in vitro and to provide two novel well-characterized acute myelomonocytic leukemia cell lines for the research of leukemia.1. Establishment and characterization of an new AML-M4 cell line (JIH-3)MethodsMost of the described human myeloid leukemia cell lines are derived from aggressive or relapsed cases and have multiple cytogenetic aberrations. There are few cell lines derived from the patients with good prognosis. Based on the experience of the extablishment of SHI-1 and SHI-2 , the primary leukemia cell from over 10 relapsed leukemia patients were cultured in vitro. The acute myelomonocytic leukemia (AML-M4) cell line ,JIH-3 was established from the peripheral blood of an AML-M4 patient. There are four parts in this chapter.In the first part, we performed comprehensive genetic characterization on the AML-M4 patient. Conventional RHG banding was emploryed for karyotypic analysis. Fluorescence in situ hybridization (FISH) was used to identify the rearrangement of CBFβgene. Chromosome painting (CP) was performed using chromosome paint probes for the short arm and long arm of chromosome 7 to identify the deletion of the short arm of chromosome 7. The specific fusion gene transcript including MYH11/ CBFβwas studied by multiplex reverse transcriptase poiymerase chain reaction (Multiplex RT-PCR).In the second part, the process of establishment of the JIH-3 cell line was described and the basic biologic features of JIH-3 were characterized. The growth feature of the JIH-3 cell line in the 24-well plate was studied under the inverted microscope. The cell line was routinely stained with Wright’s staining method. Cytochemical studies were performed byα-naphthyl butyrate esterase, and peroxidase staining. The ultrastructure was assayed by the transmitted electronic microscope. The growth curve of the JIH-3 was obtained by incubating the cells in 24-well plate and the doubling time was calculated from the curve. The clonality was assayed by semi-solid methylcellulose clonogenic assay. The immunoprofile and the cell cycle distribution of JIH-3 were studied by flow cytometry (FCM). Quantitative fluorescent polymerase chain reaction was used to detected EBV genomic DNA. DNA-PCR was performed to determine the contamination of mycoplasma. RHG banding was employed for dynamic karyotypic analysis in the process of establishment of the cell line. FISH was used to identify the rearrangement of CBFβand PML gene. In order to assess the dicentric characterization of the derivative chromosome , FISH was performed using centromere-specific probes for chromosomes 4 and 7. CP was performed using CPs for 7p and 7q. Multiple FISH was used to exclude cryptic chromosomal abnormalities. The 29 specific fusion gene transcript was studied by Multiplex RT-PCR. The exons of KIT、P53、FLT3、JAK2、NPM1、CEBPA、RUNX1 and WT1 were amplified by PCR and sequenced, respectively. Cell line authentication was performed by short tandem repeating sequences-PCR (STR-PCR). In the third part, the tumorigenicity in mice was evaluated in four nude mice subcutaneous injected with the JIH-3 cell line. The tumor specimen was studied by the conventional pathologic assay. The mononuclear cells (MNC) of the tumor was assayed by RHG-banding and FCM for the immunoprofile.In the fourth part, array comparative genomic hybridization (aCGH) was carried out to determine the genomic copy number changes (loss or gain) in primary leukemia cell from the AML-M4 patient and JIH-3 cell line. Genomic DNA was extracted from primary leukemia cell and JIH-3 cell line. aCGH study was done by the limited Bio-tech company of KangChen.Results1. The cytogenetic analysis of the patient’bone marrow (BM) cells at presentation was normal male karyotype: 46, XY. The karyotype was 46,XY, del(7)(p1?3p2?2)[10] and 46, XY, del(7)(p1?3p2?2) [10] / 46,XY[2] at the first and second relapse ,respectively. The deletion of the short arm of chromosome 7 was confirmed by CP using CPs for 7p and 7q. The rearrangement of CBFβgene was exclude by FISH. It showed negative results of rearrangement of 29 fusion genes (including MYH11/ CBFβ) by Multiplex RT-PCR. The patient’s leukemia cells showed myeloid and monocytic antigens. The diagnosis of M4eo was excluded.2. The JIH-3 cell line could be cultured in IMDM medium with 15% fetal calf serum (FBS) without any cytokine, and has been maintained in continuous culture for more than one year. It grows as single cells in suspension in the 24-well plate. JIH-3 cells remain stable proliferation after repeated freezing and thawing and has doubling time of about 102.5 hours. Most of the cells showed a regular cell shape, intermediate size, folded nucleus with prominent nucleoli, finely reticular chromatin, and blue-grey cytoplasm with azurophilic granules. Transmission electron microscopy examination showed that most of cells have numerous mitochondria, endoplasic reticulum, lysosome, vacuole and folded nucleus with prominent nucleoli. Eighteen percent of JIH-3 cells were positive for peroxide staining. The cell line was negative forα-naphthyl butyrate esterase and the NaF inhibition test. The results of immunoprofile showed that the JIH-3 cell line expressed myeloid and monocytic antigens. A few markers of T、B lymphocytic lineage、stem cell and natural killer (NK)-related antigens were also presented. Cell cycle analysis showed 17.065% of cells in S phase, 78.4% in G1 phase and 4.5% in G2 phase. The karyotype of the JIH-3 cells harvested on September 18 of 2009 revealed the presence of two clones: 46,XY,del(7p)[6]/ 45,XY,dic(4;7)(p11;p11),del(15q)[4] and the karyotype of JIH-3 cells harvested on December 22 of 2009 was 45,dic(4;7)(p11;p11),del(15q). The dicentric characterization of the derivative chromosome dic(4;7) was revealed by FISH using centromere-specific probes for chromosomes 4 and 7. The translocation between chromosomes 4 and chromosomes 7 were detected by M-FISH. The JIH-3 had no the rearrangemenst of CBFβand PML gene. The clonality of the JIH-3 cell line was confirmed by semi-solid methylcellulose clonogenic culture. EBV genomic DNA and mycoplasma contamination were not revealed. Any positive results of fusion genes were not revealed by Multiplex RT-PCR. JIH-3 had no gene mutation of KIT、P53、FLT3、JAK2、NPM1、CEBPA、RUNX1 and WT1. Cell line authentication by STR-PCR showed that the primary leukemia cells of the patient and the JIH-3 cell line originated from the same individual.3. The tumor masses were found in hypodermic tissue in all four nude mice injected with JIH-3 cells after 60 days. The sizes of the tumor masses range from 0.4×0.3～1.5×1.0cm~2. The results of the conventional pathologic assay showed the tumor masses were composed of the leukemia cells in which blood vessels and necrosis could be seen. Chromosome analysis was performed on mononuclear cells isolated from the tumor masses and showed the same chromosomal abnormalities as the JIH-3 cell line: 45,XY,dic(4;7)(p11;p11),del(15q). Their immunophenotypic profile corresponded to the JIH-3 cell line.4. Array CGH study was performed using DNA extracted from the primary leukemia cells. The aCGH analysis confirmed the deletion of the short arm of chromosome 7 identified by RHG-banding. However, the revised lost locus was 7p14.1p21.1 and 7p22.3 rather than 7p1?3p2?. The size was 18.8Mb and 601Kb, respectively. In addition to the large-scale chromosome aberrations, aCGH revealed cryptic genomic gains and losses not visible by karyotype. In JIH-3, the range of deletion was from 7p11.1 to 7p22.3(53Mb). The regions of genomic copy number changes harbor genes that maybe play an important role in the development and progression of AML. 119 of 138 genes and 302 of 366 genes were in regions of genomic copy number changes on 7p in primary leukemia cells and in JIH-3, respectively.Conclusions1. To report an acute myelomonocytic leukemia (AML-M4) patient with del(7)(p13p22) in the first relapse. The comprehensive genetic studies were performed using RHG-banding, FISH, multiplex RT-PCR and CP. The results of these studies disclosed this case to be an acute myelomonocytic leukemia with del(7p).2. A human acute myelomonocytic leukemia cell line, JIH-3, was established from an AML-M4 patient. The morphology and immunoprofile of the cell line presented typical features of myelocytic and monocytic lineage. The results of RHG-banding and CP confirmed the existence of del(7p). No rearrangement of CBFβand PML gene were disclosed by FISH. No cryptic rearrangement and fusion genes were revealed by M-FISH and multiplex RT-PCR. No gene mutation of KIT、P53、FLT3、JAK2、NPM1、CEBPA、RUNX1 and WT1 were found by sequencing of the PCR products. EBV genomic DNA and mycoplasma contamination were not found. The clonality of the JIH-3 cell line was confirmed by semi-solid methylcellulose clonogeneic culture. Cell line authentication by STR-PCR showed that the primary leukemia cells of patient and JIH-3 cell line originate from the same individual.3. The JIH-3 cell line has high tumorigenicity in nude mice.4. Array CGH is a powerful technique for detection of genomic copy number changes. The deletion of the short arm of chromosome 7 was confirmed and the lost locus was revised to 7p14.1p21.1 and 7p22.3 in primary leukemia cells. The del(7)(p11.1 p22.3) was also confirmed in JIH-3 by aCGH. And cryptic genomic gains and losses not visible by karyotype were revealed by aCGH. 2. Establishment and characterization of an new CML-BC cell line (JIH-6) MethodsThe chronic myeloid leukemia(CML)- derived leukemia cell line, JIH-6 was established from the peripheral blood of a patient in myeloid blast crisis of CML. There are two parts in this chapter.In the first part, we performed comprehensive genetic characterization on the CML patient in myeloid blast crisis. Conventional RHG banding was emploryed for karyotypic analysis. Fluorescence in situ hybridization (FISH) was used to detect the rearrangement of BCR/ABL fusion gene, AML1 and EVI1 genes. the BCR/ABL fusion transcript was detected by reverse transcriptase poiymerase chain reaction (RT-PCR). The fusion transcript of BCR/ABL gene was amplified by PCR and sequenced to exclude the mutation. The immunoprofile were studied by flow cytometry (FCM). The specimens used in all studies were from peripheral blood.In the second part, the process of establishment of the JIH-3 cell line was described and the basic biologic features of JIH-6 were characterized. The growth feature of the JIH-6 cell line in the 24-well plate was studied under the inverted microscope. The cell line was routinely stained with Wright’s staining method. Cytochemical studies were performed by peroxidase staining. The growth curve of the JIH-6 was obtained by incubating the cells in 24-well plate and the doubling time was calculated from the curve. The immunoprofile and the cell cycle distribution of JIH-6 were studied by flow cytometry (FCM). Quantitative fluorescent polymerase chain reaction was used to detect EBV genomic DNA. DNA-PCR was performed to determine the contamination of mycoplasma. RHG banding was employed for karyotypic analysis in the process of establishment of the cell line. FISH was used to identify the rearrangement of BCR/ABL fusion gene、AML1 and EVI1 gene. the BCR/ABL fusion transcript was detected by RT-PCR. The fusion transcript of BCR/ABL gene was amplified by PCR and sequenced to exclude the mutation. MDS1-EVI1/EVI1 gene expression was revealed by quantitative real-time RT-PCR in JIH-6、JIH-3、JIH-4、SHI-1、NB4 and HL-60 cell lines. Cell line authentication was performed by short tandem repeating sequences-PCR (STR-PCR).Results1. The cytogenetic analysis of the CML patient’bone marrow (BM) cells and peripheral blood showed 46, XX, t(9;22) at presentation and 46,XX, der(3)?inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11) in myeloid blast crisis, respectively. The BCR/ABL fusion gene was confirmed by FISH. The inversion of chromosome 3 was confirmed by FISH using dual-color EVI1 gene probe and EVI1 gene amplification was showed in some cells. The rearrangement of AML1 gene resulted from the translocation between chromosome 4 and 21 was cinfirmed by FISH using the probes of AML1/ETO and AML1, respectively. AML1 gene amplification was also showed in some cells. At the time of blast crisis, the BCR/ABL/ABL ratio was 12301/10000abl. A point mutation of T G of the ABL gene was found by sequencing of the PCR products. A leukemia marker study from peripheral blasts demonstrated that the blasts expressed antigens of myeloid and monocytic lineage. The diagnosis of CML in myelomonocytic blast crisis was made.2. The JIH-6 cell line could be cultured in IMDM medium with 15% fetal calf serum (FBS) without any cytokine, and has been maintained in continuous culture for more than eight months. It grows as single cells in suspension in the 24-well plate. JIH-6 cells remain stable proliferation after repeated freezing and thawing and has doubling time of about 118.2 hours. Most of the cells showed a regular cell shape, intermediate size, folded nucleus with prominent nucleoli, finely reticular chromatin, and blue-grey cytoplasm with azurophilic granules. JIH-6 cells were positive for peroxide staining. The results of immunoprofile showed that the JIH-6 cell line expressed myeloid and monocytic antigens. A few markers of T、B lymphocytic lineage and stem cell were also presented. Cell cycle analysis showed 24.78% of cells in S phase, 73.29% in G1 phase and 1.92% in G2 phase. JIH-6 showed a slow proliferation. And the karyotypic analysis only was performed on January 5 of 2010 and the result was 46,XX, inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11). Thus, the chromosome abnormalities of JIH-6 were the same as the patient’s leukemia cells. EBV genomic DNA and mycoplasma contamination were not revealed. The rearrangement and amplification of EVI1 gene were confirmed by FISH using dual-color EVI1 probe. The rearrangement and amplification of AML1 gene were also confirmed by FISH using AML1/ETO and AML1 dual-color probe. BCR/ABL fusion gene was detected by FISH and RT-PCR, respectively. The overexpression of EVI1 gene was revealed by quantitative real-time RT-PCR in JIH-6、JIH-3、JIH-4、SHI-1、NB4 and HL-60 cell lines.. Cell line authentication by STR-PCR showed that the primary leukemia cells of the patient and the JIH-6 cell line originated from the same individual.Conclusions1. To report an chronic myeloid leukemia (CML) patient in myelomonocytic blast crisis with inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11). The comprehensive genetic studies were performed using RHG-banding, FISH, RT-PCR and sequencing of PCR products. The results of these studies disclosed this case to be an CML in myelomonocytic blast crisis with a t(9;22) which resulted in BCR/ABL rearrangement, inv(3)(q21q26) which resulted in EVI1 gene rearrangement and amplification, and t(4;21) which resulted in AML1 gene rearrangement and amplification and a point mutation of ABL gene. Both are associated with her poor prognosis.2. A human CML with t(9;22) cell line, JIH-6, was established from an CML patient in myelomonocytic blast crisis. The morphology and immunoprofile of the cell line presented typical features of myelomonocytic lineage. The results of RHG-banding showed the existence of inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11). The rearrangement of BCR/ABL were disclosed by FISH and PCR. The rearrangement and amplification of EVI1 and AML1 genes were revealed by FISH. No gene mutation of ABL were found by sequencing of the PCR products. EBV genomic DNA and mycoplasma contamination were not found. Cell line authentication by STR-PCR showed that the primary leukemia cells of patient and JIH-6 cell line originated from the same individual.In summary, JIH-3 and JIH-6 are two new myelomonocytic leukemia cell lines. the karyotype of JIH-3 is del(7p), and the JIH-6 is t(9;22), inv(3)(q21q26) and t(4;21)(q21;q22). They have clear biologic background and provides two new important tools for leukemia research.