Hematopoietic- and neurologic-expressed sequence 1 expression in the murine GL261 and high-grade human gliomas

Abstract

The hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a highly conserved protein that is expressed in developing and regenerating tissues. In this study, Hn1 expression was evaluated in human and murine malignant gliomas. Hn1 mRNA and protein were detected in the murine GL261 glioma cell line and in GL261 brain tumors in vivo. HN1 is also expressed in human U118MG and U87MG cell lines. Evaluation of human brain tumors using an anti-Hn1 polyclonal antibody detected strong immunoreactivity in high-grade (WHO III and IV) malignant gliomas. The rate of GL261 cell proliferation in vitro was unaltered by Hn1 depletion using an anti-Hn1 siRNA. However, tumors established from Hn1-depleted GL261 cells formed significantly smaller volumes than those established from control-treated cells. These data suggest a role for Hn1 in the biology of malignant brain tumors.

Fig. 1

Hn1 is expressed in murine and human glioma cell lines. (a) Northern blot analysis of Hn1 mRNA in GL261 cells. The migration of the 18 S rRNA is indicated. (b) Western blot analysis of HEK293 cells (HEK), HEK293 cells transfected with a recombinant murine Hn1 protein (HEK-Hn1), murine B16.F10 and GL261 cells, and the human U118 and U87 glioma cells using the rabbit polyclonal anti-Hn1 antibody. (c) Western blot analysis, using the anti-Hn1 antibody, show Hn1 expression in GL261 cells transduced at an M.O.I. of 5,000 with either Hn1-siRNA AAV6 (siRNA) or control H1-AAV6 (H1-control)

Fig. 2

Hn1 is expressed in GL261 brain tumors of C57BL/6 mice. Sections were derived from tumor bearing brains 20 days post-GL261 cell implantation. Panels a–b depict low resolution ISH analysis of Hn1 mRNA in GL261 tumors using anti-sense (a) and sense (b) Hn1 riboprobes. Panels (c–d) depict higher resolution silver grain analysis of adjacent sections from a GL261 brain tumor hybridized with anti-sense (c) and sense (d) Hn1 riboprobes. High silver grain density on the left side of the section indicates Hn1 expression within the tumor cells. Sections were counterstained with H&E following ISH. (e) Immunohistochemical analysis using a rabbit polyclonal anti-Hn1 antibody showing Hn1 (red) in the GL261 tumor cells and not the GFP-expressing microglia (green). Sections were counterstained with DAPI nuclear stain (blue)

Fig. 3

HN1 is expressed in multiple types of high-grade human glioma. A human brain tumor tissue microarray containing a variety of tumor samples was subjected to immunohistochemistry using the rabbit polyclonal anti-Hn1 antibody. Strong immunoreactivity was identified in human malignant gliomas including glioblastoma multiforme (a–c), anaplastic oligodendroglioma (d), and anaplastic astrocytoma (e); panel (f) shows a healthy region of brain cortex

Fig. 4

Hn1 depletion affects growth of GL261 tumors in vivo but not in vitro proliferation rates of GL261 cells. (a) In vitro growth rates of GL261 cells transduced with either Hn1-siRNA AAV6 (grey), control H1 AAV6 (black) at an M.O.I. of 5,000, or no virus (white). There was no difference in the in vitro growth rates of GL261 cells with or without Hn1. (b) Comparison of the growth of in vivo GL261 tumors established from cells pretreated with either control or Hn1-siRNA-expressing AAV6. The scatter-plot shows individual tumor volumes as well as means and SEMs. The difference between the mean volumes of tumors derived from Hn1-expressing and Hn1-depleted cells was statistically significant (p<0.05)