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Cervical cancer is a leading cause of cancer-‐related death among women. The SiHa cell, a cervical cancer cell line, expresses kera>n 8/18 (K8/18) intermediate filaments that when elevated is indica>ve of neoplas>c changes. The kera>n 18 monomer of kera>n filaments is glycosylated by N-‐acetylglucosmine (O-‐GlcNAc). In general, hyper-‐O-‐GlcNAcyla>on is a common characteris>c of many cancers. However, poten>al connec>ons between O-‐GlcNAcyla>on, K8/18 filament expression, and the tumorigenic poten>al of cells expressing these traits have not been explored. Here we tested the hypothesis that O-‐GlcNAcyla>on in SiHa cells promotes tumorigenic poten>al. Accordingly, growth experiments indicated that SiHa cells grown under controlled culture condi>ons mul>plied faster than cells exposed to Alloxan, an O-‐GlcNAcyla>on inhibitor (0.31 vs. 0.08 doublings/day, respec>vely), and had a shorter genera>on >me (2.27 vs. 8.78 days, respec>vely; n= 3 experiments). A wound-‐healing assay demonstrated that SiHa cells migrate faster aSer injury vs. cells exposed to Alloxan (5.5 vs. 4.1 µm/hour, respec>vely; n=3 experiments). These ini>al observa>ons suggest that O-‐GlcNAcyla>on in SiHa cells, possibly via K8/18 filaments, increases tumorigenic poten>al. The results provide further insight about the poten>al influence of glycosyla>on and kera>n filaments on cell physiology beyond simply being a diagnos>c measure of cancer.
An es>mated half-‐million new cases of cervical cancer are recorded annually worldwide, half of which result in death. A connec>on between infec>on with the human papillomavirus (HPV) and the onset of cervical cancer was discovered two decades ago. More than 99% of cervical cancers are caused by the high-‐risk HPV viruses (HPV) 16 and 18 [1]. The HPV16-‐infected cervical cancer cell line (SiHa) was selected as a model to inves>gate aspects of tumorigenesis. Specifically, we inves>gated the role of glycosyla>on by N-‐acetylglucosmine (O-‐GlcNAc) as a poten>al influence on tumorigenicity. Global O-‐GlcNAcyla>on was manipulated with Alloxan, an O-‐GlcNAc transferase (OGT) inhibitor, and PUGNAc, an O-‐GlcNAcase inhibitor (OGA) [2].
INTRODUCTION
HYPOTHESIS
Cell Prolifera6on Assay • Treatment groups included cells cultured in the absence (Control) and presence of 5mM Alloxan
• Flat-‐bocomed growth tubes were seeded with 100K SiHa cells in EMEM, 10% FBS, with an>bio>cs
• Condi>oned medium and treatments were exchanged daily, and cultures from each treatment group were harvested for coun>ng every 24 hours
Cell Migra6on Assay • Treatment groups included cells cultured in the absence (Control) and presence of 5mM Alloxan
• Confluent monolayers of SiHa cells in 24 well plates were “wounded” with a cell scraper
• Wound healing was imaged and the percent closure and cell migra>on rate were calculated every 24 hours
Cell Invasion Assay • Treatment groups included cells cultured in the absence (Control) and presence of either 5mM Alloxan or 200mM PUGNAc
• 100K SiHa cells were seeded into 12 well Transwell inserts having 12μm pores (Corning; Corning, NY)
• FBS was used as a chemoacractant in the lower chamber, and the cultures were incubated 24 hours. Inserts were then fixed, stained with Crystal Violet, and imaged before being destained in 70% ethanol to measure absorbance at 540nm
MATERIALS & METHODS
RESULTS Hypo-‐O-‐GlcNAcyla-on Impairs Cell Prolifera-on
O-‐GlcNAcyla>on promotes tumorigenic poten>al of cervical cancer cells.
Figure 1. Inhibi.ng O-‐ GlcNAcyla.on in SiHa cells (via Alloxan) prevented cell prolifera.on (p<0.0001; n=3 independent experiments). There was no effect, however, on cell viability.
RESULTS (CONTINUED)
Figure 2. Representa.ve images from three independent experiments. Inhibi.on of O-‐GlcNAcyla.on reduced the cell migra.on rate (P<0.001), and thus the wound healing poten.al of SiHa cells. DoRed red lines indicate loca.on of the ini.al wound. Photos at 40x magnifica.on.
RESULTS (CONTINUED)
Figure 4.Graph is representa.ve of cell migra.on from 3 independent migra.on invasion experiments in which migra.on was only impaired by inhibi.on of O-‐GlcNAcyla.on (Alloxan), (P<0.01).
SUMMARY • Inhibi>on O-‐GlcNAcyla>on impaired SiHa cell
prolifera>on (Figure 1), cell migra>on (Figure 2), and cell invasion (Figures 3 and 4).
• Hyper-‐O-‐GlcNAcyla>on had no effect on cell invasion (Figure 4).
REFERNCES
ACKNOWLEDGMENTS & CONTACT
Stephanie Parisi stg27@wildcats.unh.edu University of New Hampshire
This material is based upon work supported by the COLSA Karabelas Fund. Par>cular apprecia>on is expressed to Dr. Townson and Dr. Foxall and their laboratories for providing materials and assistance with the experiments.
Hypo-‐O-‐GlcNAcyla-on Inhibits Cell Migra-on
Hypo-‐O-‐GlcNAcyla-on Prevents Cell Invasion; Hyper-‐O-‐GlycNacyla-on Has No Effect
David H. Towson dave.townson@unh.edu University of New Hampshire Kendall Hall Room 509 Durham, NH 03824
Total number of genera>ons: 2.2 vs. 0.6 (Control vs. Alloxan) Mul>plica>on rate: 0.4 vs. 0.1 doublings per day Genera>on >me: 2.3 vs. 7.8 days
1. Liao S, Lee W, Chen H, Chuang L, Pan M, Chen C. Baseline human papillomavirus infec>on, high vaginal parity, and their interac>on on cervical cancer risks aSer a follow-‐up of more than 10 years. Cancer Causes & Control 2012(5):703
2. Varki, A. et al. Essen>als of Glycobiology What is Glycobiology ?. (Cold Spring Harbor Laboratory Press, 2009).
Figure 3. Representa.ve images from three independent cell invasion experiments. Crystal Violet-‐stained cells that have migrated through the Transwell insert pores are shown. Photos at 40x magnifica.on.
ABSTRACT
T=0 hrs 0% Migra6on
T=48 hrs 40% Migra6on
T=96 hrs 92% Migra6on
Control
T=0 hrs 0% Migra6on
T=48 hrs 3% Migra6on
T=96 hrs 60% Migra6on
Hypo-‐O-‐GlcNAcyla6on
CONCLUSION O-‐GlcNAcyla>on enhances tumorigenesis
of cervical cancer cells.
OH
K8/18 Intermediate Filaments
GlcNAc UDP-‐GlcNAc UDP
O-‐GlcNAcase (OGA)
O-‐GlcNAc transferase (OGT)
GlcNAc H20
Chemoa[ractant Present
No Chemoa[ractant Present
Hypo-‐O-‐GlcNAcyla6on
Hyper-‐O-‐GlcNAcyla6on Control
Control
Blank Insert No Cells
Hypo-‐O-‐GlcNAcyla6on
Hyper-‐O-‐GlcNAcyla6on
Control Hypo-‐O-‐GlcNAcyla6on
Migrated Surface Area 5mm2 4mm2
Migra>on Rate 5.5μm /hour
4.1μm/hour
Full wound closure 120 hours 168 hours
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
Control Hyper-‐O-‐GlcNAcyla>on
Hypo-‐O-‐GlcNAcyla>on
Rela6v
e Ab
sorban
ce (5
40nm
)
Chemoacractant Present No Chemoacractant Present
a b b
b
b
PUGNAc
Alloxan
a