Sister chromatid cohesion defects are associated with chromosome instability in Hodgkin lymphoma cells

Abstract

Background: Chromosome instability manifests as an abnormal chromosome complement and is a pathogenic event in cancer. Although a correlation between abnormal chromosome numbers and cancer exist, the underlying mechanisms that cause chromosome instability are poorly understood. Recent data suggests that aberrant sister chromatid cohesion causes chromosome instability and thus contributes to the development of cancer. Cohesion normally functions by tethering nascently synthesized chromatids together to prevent premature segregation and thus chromosome instability. Although the prevalence of aberrant cohesion has been reported for some solid tumors, its prevalence within liquid tumors is unknown. Consequently, the current study was undertaken to evaluate aberrant cohesion within Hodgkin lymphoma, a lymphoid malignancy that frequently exhibits chromosome instability.

Methods: Using established cytogenetic techniques, the prevalence of chromosome instability and aberrant cohesion was examined within mitotic spreads generated from five commonly employed Hodgkin lymphoma cell lines (L-1236, KM-H2, L-428, L-540 and HDLM-2) and a lymphocyte control. Indirect immunofluorescence and Western blot analyses were performed to evaluate the localization and expression of six critical proteins involved in the regulation of sister chromatid cohesion.

Results: We first confirmed that all five Hodgkin lymphoma cell lines exhibited chromosome instability relative to the lymphocyte control. We then determined that each Hodgkin lymphoma cell line exhibited cohesion defects that were subsequently classified into mild, moderate or severe categories. Surprisingly, ~50% of the mitotic spreads generated from L-540 and HDLM-2 harbored cohesion defects. To gain mechanistic insight into the underlying cause of the aberrant cohesion we examined the localization and expression of six critical proteins involved in cohesion. Although all proteins produced the expected nuclear localization pattern, striking differences in RAD21 expression was observed: RAD21 expression was lowest in L-540 and highest within HDLM-2.

Conclusion: We conclude that aberrant cohesion is a common feature of all five Hodgkin lymphoma cell lines evaluated. We further conclude that aberrant RAD21 expression is a strong candidate to underlie aberrant cohesion, chromosome instability and contribute to the development of the disease. Our findings support a growing body of evidence suggesting that cohesion defects and aberrant RAD21 expression are pathogenic events that contribute to tumor development.

Figure 1

HL cell lines exhibit cohesion defects. (A) Representative images of mitotic chromosome spreads from the L-540 cells demonstrating normal primary constriction cohesion (Upper left) and the various categories of aberrant cohesion; PCG_I (Mild), PCG_II (Moderate), and PCG_III (Severe). Each cohesion category (quadrant) contains a low-resolution DAPI image of the entire mitotic spread. Each spread contains a white bounding box that defines the region magnified and presented on the right-hand side of each quadrant. Scale bar represents 10 μm. (B) Graphs depicting the total fraction of cells with aberrant cohesion; white (normal), black (aberrant). The total number of mitotic spreads evaluated for each cell line is indicated at the bottom of each column. (C) Graphical depiction for only the fraction of mitotic spreads with cohesion defects. The frequencies of defects are classified according to three different aberrant cohesion categories (e.g. PCG_I - light gray; PCG_II - dark gray; and PCG_III - black). The total number of mitotic spreads exhibiting aberrant cohesion is indicated at the base of each column.

Figure 2

Spatial localization of cohesion-related proteins in L-540 cells. Indirect immunofluorescence was employed to determine the spatial localization of six proteins involved in sister chromatid cohesion (SMC1A, SMC3, STAG2, RAD21, Securin and Separase). Presented are representative high-resolution (63×) images obtained from L-540 cells in interphase. Nuclei were counterstained with DAPI and immunofluorescently labeled for the epitope indicated on the left. Scale bar represents 3 μm.

Figure 3

Expression levels of cohesion-related proteins in HL cell lines. Representative immunoblots depicting the relative expression levels of 6 specific cohesion-related proteins in a lymphocyte control and five HL cell lines (indicated at top) with α-tubulin serving as a loading control. Semi-quantitative values for each cohesion epitope were determined and adjusted for unequal loading by generating a ratio with corresponding loading control. To further facilitate comparisons between cell lines, all semi-quantitative values were normalized to the lymphocyte control (1^st lane in each gel; values set to 1.0) with the relative expression levels indicated.