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Establishment and characterization of two primary breast cancer cell lines from young Indian breast cancer patients: mutation analysis.

Pandrangi SL, Raju Bagadi SA, Sinha NK, Kumar M, Dada R, Lakhanpal M, Soni A, Malvia S, Simon S, Chintamani C, Mohil RS, Bhatnagar D, Saxena S - Cancer Cell Int. (2014)

Bottom Line: Karyotype analysis showed aneuploidy, deletions and multiple rearrangements in chromosomes 7, 9, X and 11 and isochromosomes 17q in both the cell lines.P53 mutational analysis revealed no mutation in the coding region in both the cell lines; however NIPBC-2 cell line showed presence of heterozygous C/G polymorphism, g.417 C > G (NM_000546.5) resulting in Arg/Pro allele at codon 72 of exon 4.Screening for mutations in BRCA1&2 genes revealed presence of three heterozygous polymorphisms in exon 11 of BRCA1 and 2 polymorphisms in exons 11, and14 of BRCA2 gene in both the cell lines.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi 110029, India. sunita_saxena@yahoo.com.

ABSTRACT
Two novel triple negative breast cancer cell lines, NIPBC-1 and NIPBC-2 were successfully established from primary tumors of two young breast cancer patients aged 39 and 38 years respectively, diagnosed as infiltrating duct carcinoma of breast. Characterization of these cell lines showed luminal origin with expression of epithelial specific antigen and cytokeratin 18 and presence of microfilaments and secretary vesicles, microvilli, tight junctions and desmosomes on ultra-structural analysis. Both the cell lines showed anchorage independent growth and invasion of matrigel coated membranes. Karyotype analysis showed aneuploidy, deletions and multiple rearrangements in chromosomes 7, 9, X and 11 and isochromosomes 17q in both the cell lines. P53 mutational analysis revealed no mutation in the coding region in both the cell lines; however NIPBC-2 cell line showed presence of heterozygous C/G polymorphism, g.417 C > G (NM_000546.5) resulting in Arg/Pro allele at codon 72 of exon 4. Screening for mutations in BRCA1&2 genes revealed presence of three heterozygous polymorphisms in exon 11 of BRCA1 and 2 polymorphisms in exons 11, and14 of BRCA2 gene in both the cell lines. Both the cell lines showed presence of CD 44+/24-breast cancer stem cells and capability of producing mammosphere on culture. The two triple negative breast cancer cell lines established from early onset breast tumors can serve as novel invitro models to study mechanisms underlying breast tumorigenesis in younger age group patients and also identification of new therapeutic modalities targeting cancer stem cells.

No MeSH data available.


Related in: MedlinePlus

Establishment of NIPBC-1 cell line showing stages of (a) Initiation (b) purification and (c) completely purified confluent NIPBC-1 cell line.
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Figure 1: Establishment of NIPBC-1 cell line showing stages of (a) Initiation (b) purification and (c) completely purified confluent NIPBC-1 cell line.

Mentions: We have initiated 31 primary cultures using 44 biopsies obtained from patients diagnosed with carcinoma of breast. Out of these primary cultures, 2 cell lines could be successfully purified and propagated for more than 60 passages. Among the two cell lines established, NIPBC-1 was derived from a breast cancer patient aged 39 years and NIPBC-2 from a 38 year old patient. The breast tumor in both the cases has been diagnosed as Infiltrating breast cancer NOS type, grade IIb and grade III respectively. The two cell lines were established by enzymatic disaggregation followed by differential trypsinization. NIPBC-1 was initiated after enzymatic disaggregation of tumor tissue with trypsin. Cells were seeded onto the surface of a flask and cells which adhered to the surface by day 1, reached confluency by day 10; the cells were then subjected to differential trypsinization, until a pure epithelial cell population is remained in the flask. NIPBC-1 cells are spindle shaped cells which grow sparse, do not grow in layers and adhere strongly to the surface (Figure 1). The second cell line NIPBC-2 was initiated by enzymatic disaggregation with both trypsin and collagenase; the cells adhered to the surface after day 3, and formed cell aggregates consisting of epithelioid cells. These cells proliferated and occupied the whole surface by day 8. They formed distinct large epithelial colonies surrounded by fibroblasts, which were further enriched by selective scraping of the fibroblasts. NIPBC-2 cells are cuboidal cells which form multilayers. They are fast growing, and easily get detached upon trypsinization. NIPBC-1 and NIPBC-2 cell lines were so far passaged for 85 and 66 times respectively in our laboratory (Figure 2).


Establishment and characterization of two primary breast cancer cell lines from young Indian breast cancer patients: mutation analysis.

Pandrangi SL, Raju Bagadi SA, Sinha NK, Kumar M, Dada R, Lakhanpal M, Soni A, Malvia S, Simon S, Chintamani C, Mohil RS, Bhatnagar D, Saxena S - Cancer Cell Int. (2014)

Establishment of NIPBC-1 cell line showing stages of (a) Initiation (b) purification and (c) completely purified confluent NIPBC-1 cell line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016554&req=5

Figure 1: Establishment of NIPBC-1 cell line showing stages of (a) Initiation (b) purification and (c) completely purified confluent NIPBC-1 cell line.
Mentions: We have initiated 31 primary cultures using 44 biopsies obtained from patients diagnosed with carcinoma of breast. Out of these primary cultures, 2 cell lines could be successfully purified and propagated for more than 60 passages. Among the two cell lines established, NIPBC-1 was derived from a breast cancer patient aged 39 years and NIPBC-2 from a 38 year old patient. The breast tumor in both the cases has been diagnosed as Infiltrating breast cancer NOS type, grade IIb and grade III respectively. The two cell lines were established by enzymatic disaggregation followed by differential trypsinization. NIPBC-1 was initiated after enzymatic disaggregation of tumor tissue with trypsin. Cells were seeded onto the surface of a flask and cells which adhered to the surface by day 1, reached confluency by day 10; the cells were then subjected to differential trypsinization, until a pure epithelial cell population is remained in the flask. NIPBC-1 cells are spindle shaped cells which grow sparse, do not grow in layers and adhere strongly to the surface (Figure 1). The second cell line NIPBC-2 was initiated by enzymatic disaggregation with both trypsin and collagenase; the cells adhered to the surface after day 3, and formed cell aggregates consisting of epithelioid cells. These cells proliferated and occupied the whole surface by day 8. They formed distinct large epithelial colonies surrounded by fibroblasts, which were further enriched by selective scraping of the fibroblasts. NIPBC-2 cells are cuboidal cells which form multilayers. They are fast growing, and easily get detached upon trypsinization. NIPBC-1 and NIPBC-2 cell lines were so far passaged for 85 and 66 times respectively in our laboratory (Figure 2).

Bottom Line: Karyotype analysis showed aneuploidy, deletions and multiple rearrangements in chromosomes 7, 9, X and 11 and isochromosomes 17q in both the cell lines.P53 mutational analysis revealed no mutation in the coding region in both the cell lines; however NIPBC-2 cell line showed presence of heterozygous C/G polymorphism, g.417 C > G (NM_000546.5) resulting in Arg/Pro allele at codon 72 of exon 4.Screening for mutations in BRCA1&2 genes revealed presence of three heterozygous polymorphisms in exon 11 of BRCA1 and 2 polymorphisms in exons 11, and14 of BRCA2 gene in both the cell lines.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi 110029, India. sunita_saxena@yahoo.com.

ABSTRACT
Two novel triple negative breast cancer cell lines, NIPBC-1 and NIPBC-2 were successfully established from primary tumors of two young breast cancer patients aged 39 and 38 years respectively, diagnosed as infiltrating duct carcinoma of breast. Characterization of these cell lines showed luminal origin with expression of epithelial specific antigen and cytokeratin 18 and presence of microfilaments and secretary vesicles, microvilli, tight junctions and desmosomes on ultra-structural analysis. Both the cell lines showed anchorage independent growth and invasion of matrigel coated membranes. Karyotype analysis showed aneuploidy, deletions and multiple rearrangements in chromosomes 7, 9, X and 11 and isochromosomes 17q in both the cell lines. P53 mutational analysis revealed no mutation in the coding region in both the cell lines; however NIPBC-2 cell line showed presence of heterozygous C/G polymorphism, g.417 C > G (NM_000546.5) resulting in Arg/Pro allele at codon 72 of exon 4. Screening for mutations in BRCA1&2 genes revealed presence of three heterozygous polymorphisms in exon 11 of BRCA1 and 2 polymorphisms in exons 11, and14 of BRCA2 gene in both the cell lines. Both the cell lines showed presence of CD 44+/24-breast cancer stem cells and capability of producing mammosphere on culture. The two triple negative breast cancer cell lines established from early onset breast tumors can serve as novel invitro models to study mechanisms underlying breast tumorigenesis in younger age group patients and also identification of new therapeutic modalities targeting cancer stem cells.

No MeSH data available.


Related in: MedlinePlus