Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Jan;108(1):89-97.
doi: 10.1046/j.1365-2567.2003.01559.x.

Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene

Magnus Sundström  1 , Harissios Vliagoftis, Peter Karlberg, Joseph H Butterfield, Kenneth Nilsson, Dean D Metcalfe, Gunnar Nilsson
Affiliations

Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene

Magnus Sundström et al. Immunology. 2003 Jan.

Abstract

The human mast cell line (HMC)-1 cell line is growth-factor independent because of a constitutive activity of the receptor tyrosine kinase Kit. Such deregulated Kit activity has also been suggested causative in gastrointestinal stromal tumours (GISTs) and mastocytosis. HMC-1 is the only established continuously growing human mast cell line and has therefore been widely employed for in vitro studies of human mast cell biology. In this paper we describe two sublines of HMC-1, named HMC-1(560 ) and HMC-1(560,816 ), with different phenotypes and designated by the locations of specific mutations in the c-kit proto-oncogene. Activating mutations in the Kit receptor were characterized using the pyrosequencing trade mark method. Both sublines have a heterozygous T to G mutation at codon 560 in the juxtamembrane region of the c-kit gene causing an amino acid substitution of Gly-560 for Val. In contrast, only HMC-1(560,816) cells have the c-kitV816 mutation found in mast cell neoplasms causing an Asp-->Val substitution in the intracellular kinase domain. Kit was constitutively phosphorylated on tyrosine residues and associated with phosphatidylinositol 3'-kinase (PI 3-kinase) in both variants of HMC-1, but this did not lead to a constitutive phosphorylation of Akt or extracellular regulated protein kinase (ERK), which are signalling molecules normally activated by the interaction of stem cell factor (SCF) with Kit. The documentation and characterization of two sublines of HMC-1 cells provides both information on the biological consequences of mutations in Kit and recognition of the availability of what in reality are two distinct cultured human mast cell lines.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Morphologic analysis of HMC-1 cells. HMC-1560 (a) and HMC-1560,816 cells (b) were cytocentrifuged and stained with May–Grünwald Giemsa.
Figure 2
Figure 2
Comparison of rate of proliferation for HMC-1560 and HMC-1560,816. Cells (5 × 104/ml) were seeded on day 0. Cell numbers on days 1–7 were determined using a Coulter cell counter. Relative cell number is the cell count divided by the number of seeded cells.
Figure 3
Figure 3
Flow cytometry analysis of expression of cell surface antigens on HMC-1560 and HMC-1560,816 cells. (a) The forward and light scatter of HMC-1560 cells (left) and HMC-1560,816 cells (right). Cells were stained as shown in (b) for expression of CD13, CD32, the α-chain of FcεRI, and Kit (first grouping), CD18, CD44 and CD54 (second grouping) and CD49b, CD49c and CD49f (bottom panel). Isotype-matched antibodies were used as controls.
Figure 4
Figure 4
Analysis of mutations at codon 560 (a–c) and at codon 816 (d–f). RNA from HMC-1560 cells (a,d), HMC-1560,816 cells (b,e) and CBMC (c,f) was extracted and used for RT–PCR. The PCR products were analysed for the 560 and 816 point mutations by means of pyrosequencing™. The arrows in a–c indicates the T→G substitution at position 560, and the arrows in d–f indicate the A→T substitution at position 816. E = enzyme, S = substrate.
Figure 5
Figure 5
c-kit mutations in DNA from HMC-1 variants and patients with systemic mastocytosis (SM). In c-kit, the A-to-T substitution at nucleotide 8213 of exon 17 creates a new HinfI restriction site. Genomic DNA from patients with systemic mastocytosis and from HMC-1560 and HMC-1560,816 cells was extracted and a segment of the DNA including this site was amplified by primers complimentary to the sequence of introns 16 and 17 of the c-kit gene. The PCR product was digested with HinfI and separated on polyacrylamide gel. Predicted sizes of digested fragments are 271 and 51 bp for wild type, and 257, 51 and 14 bp for mutated c-kit. Samples are: lane 1, patient 1 with indolent mastocytosis; lane 2, patient 2 with mastocytosis with an associated hematologic disorder; lane 3, ladder; lane 4, HMC-1560; and lane 5, HMC-1560 816.
Figure 6
Figure 6
Phosphorylation of Kit in HMC-1 and MO7e cells. Cells were treated with SCF (100 ng/ml) for 15 min at 37° and lysed. Kit was immunoprecipitated with a rabbit anti-human-Kit antibody and protein A-sepharose beads from the cell lysates. Immunoblot was performed with the antiphosphotyrosine mAb 4G10.
Figure 7
Figure 7
Intracellular signalling downstream of Kit in HMC-1 cells. (a) Kit immunoprecipitates were separated on a gel and analysis of association of c-Kit with PI 3-kinase was performed by immunoblotting with an antibody recognising the p85 regulatory subunit of PI 3-kinase. The effect of the PI3-kinase inhibitor Ly 29400 on HMC-1 proliferation (b) and apoptosis (c) was investigated. Open bars represent HMC-1560; and filled bars HMC-1560, 816.
Figure 8
Figure 8
Phosphorylation of Akt and ERK. Cells were treated with SCF (100 ng/ml) for 15 min and lysed. Total cell lysates were analysed for phosphorylation of Akt (a) or ERK (b). Antibodies against total AKT and ERK, respectively, were used for control of loading.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Butterfield JH, Weiler D, Dewald G, Gleich GJ. Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk Res. 1988;12:345–55. - PubMed
    1. Nilsson G, Blom T, Kusche-Gullberg M, Kjellen L, Butterfield JH, Sundstrom C, Nilsson K, Hellman L. Phenotypic characterization of the human mast-cell line HMC-1. Scand J Immunol. 1994;39:489–98. - PubMed
    1. Valent P, Bettelheim P. Cell surface structures on human basophils and mast cells: biochemical and functional characterization. Adv Immunol. 1992;52:333–423. - PubMed
    1. Irani AM, Nilsson G, Miettinen U, Craig SS, Ashman LK, Ishizaka T, Zsebo KM, Schwartz LB. Recombinant human stem cell factor stimulates differentiation of mast cells from dispersed human fetal liver cells. Blood. 1992;80:3009–21. - PubMed
    1. Li L, Macpherson JJ, Adelstein S, Bunn CL, Atkinson K, Phadke K, Krilis SA. Conditioned media from a cell strain derived from a patient with mastocytosis induces preferential development of cells that possess high affinity IgE receptors and the granule protease phenotype of mature cutaneous mast cells. J Biol Chem. 1995;270:2258–63. - PubMed

Publication types

  NODES
admin 1
Association 1
twitter 2