L428 cell morphophenotypic changes induced by CD99 overexpression via NF-κB inactivation

Abstract

Background: Classic Hodgkin’s lymphoma (HL) is characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells. CD99 downregulation produces HRS-like cells and morphological changes in B lymphoma. The NF-κB pathway protects HRS cells from apoptosis and is in part responsible for its unique functional characteristics in Hodgkin’s lymphoma.

Objective: To observe the changes in the morphological and immunophenotype characteristics of L428 cells following transformation with CD99 and explore the role of NF-κB in these changes.

Methods: A mammalian expression vector driving CD99 was constructed and transfected into the L428 cell line (L428-MVC). L428, L428-EVC (empty vector) and L428-MVC were analyzed by immunohistochemistry (IHC), immunofluorescence, and Western blot analysis, and comparisons were made between the three cell lines with regards to the expression of CD2, CD3, CD15, CD20, CD30, CD79, CD99 and NF-κBp65. In addition, cell proliferation was monitored by MTT assay, while apoptosis and cell cycle status were determined by flow cytometry.

Results: Transfection of L428 cells with CD99 altered the morphological and immunophenotypic characteristics of HRS cells. Expression of CD15, CD30, and NF-κBp65 disappeared, while CD2, CD3, CD20 and CD79 levels remained negative. In addition, the presence of CD99 led to cell cycle arrest at G2/M and a reduction in cell proliferation. More importantly, CD99 dramatically decreased cell viability and increased cellular apoptosis compared with naive L428 cells and the L428-EVC cell line. These results indicated that the upregulation of CD99 altered cell morphology and immunophenotype, while also decreasing viability in vitro.

Conclusions: CD99 may play a key role in affecting cell morphology and immunophenotype characteristics via inactivation of NF-κBp65.