In the medulla Bone Marrow, stromal cells help support hematopoietic cells in the inflammatory process.
Two important types of stromal cells involved in early cancer development, HS5, and HS27a cell lines, contribute to the study of tumor cell growth and stromal interactions by defining the dialogue between stromal and clonal cells. According to Li et al., HS5 guarantees several cytokines that promote the proliferation of progenitor cells and HS27a supports the early formation of “rock sites” in hematopoietic prognostic cells.1
Previous studies have identified more than 17,000 genes in this cell line; However, the researchers did not look at the total protein. To meet this requirement, Li et al. A stable amino acid and cell culture isotope marker (SILAC) was used to study protein differences in HS5 and HS27a cells.
The researchers inoculated the cells, using two stable isotopes each of arginine and lysine to bind the proteins to the two cell lines. They then tested the purified proteins using 2-D ultra-high-resolution liquid chromatography and an Orbitrap LTQ (Thermo Scientific) mass spectrometer. They used MaxQuant to analyze the raw data, as well as getting a false positive of 0.01 for proteins and peptides that had a peptide length of at least six amino acids. They used Swiss-Prot and the Integrated Annotation, Visualization and Discovery Database (DAVID) to classify proteins based on their biological functions.
The researchers found a total of 4,213 unique proteins in the cell lines in Bone Marrow. Of these proteins, 1,462 proteins were identified in two independent experiments. Li and his colleagues also found that 69 proteins underwent significant changes and 48 showed a significant decrease (> 95% confidence) in HS27a compared to HS5.
Reflecting on genetic ontology and pathway analysis, the team concluded that differentially controlled proteins influence cell movement, cell-cell signaling and interactions, and the development and work of the blood system. They found a total of fifty-five items in the genomics and proteomics database.
For further validation, the researchers used quantitative transcriptional polymerase chain reaction (RT-PCR) and Western blotting of seven proteins selected from 28 different proteins. This protein is also available in open source databases and agrees on most networks/paths. The proteins include HCLS1, COL1A2, ALCAM, MAP4, HSP90, and CSE1L. The team found mRNA levels in the high-density proteins HCLS1, COL1A2, and ALCAM, and HS27a, but found mRNA levels in the low-density proteins MAP4, HSP90, and CSE1L and HS27a associated with HS5. They concluded that these results are consistent with the data on protein and genome expression.
For another protein, integrin alpha-V (ITGAV), they found low cellular expression in HS27a for HS5. In the protein database, however, they found elevated levels of the HS27a protein. Support of RT-PCR and Western blotting showed increased ITGAV levels in HS27a and mRNA levels in proteins.
The team inhibited the expression of ITGAV in HS27a cells through a defined siRNA. They observed reduced apoptosis in KG1a cells after completion of HS27a mutant cells under TNF administration. The team also explained that these findings indicate that ITGAV is involved in the cultural process. Since this task is not understood, the team plans to specifically study ITGAV for bone marrow stromal cells.
It was further emphasized that this protein system is a good starting point for future studies, including biopsy and bone microenvironment and crosstalk between stromal and myeloma cells, as well as cultural systems.