The effect of hypoxia on protein phosphatase 2A (PP2A) in MDA-MB-231 cells, and, a pilot study to establish a primary breast cancer cell model from Fine Needle Aspirations.

Date
2018-03
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
Background: Cancerous tumours are characterised by areas of insufficient blood supply, with resulting hypoxia and nutrient deprivation. The mechanisms by which cancer cells adapt to these conditions are of interest due to potential clinical relevance. Although immortalised cell lines have been used extensively in cancer research, a primary cancer cell model could potentially shed additional light on cancer biology. Protein phosphatase 2A (PP2A) is a Serine/Threonine protein phosphatase which has been implicated in hypoxia, although contradictory reports regarding its involvement has been published. Aims: 1. To establish a primary cell culture model using fine needle aspiration (FNA) for cell collection. 2. To investigate the effect of hypoxia on these primary cells. 3. To investigate the effect of hypoxia on PP2A expression, post-translational modification and activity in MDA-MB-231 cells. We hypothesized that hypoxia would induce a reduction in PP2A activity, thereby favouring the phosphorylation and activation of enzymes associated with survival and proliferation. Methods: Nine FNA samples were collected from patients at Tygerberg Hospital. Samples were subjected to processing and cultured at 37°C in suitable growth medium containing HEPES and epidermal growth factor. Cells were maintained in culture for 1.5–6 weeks and medium was refreshed initially after 3-4 days, and there after every second day. Although cells appeared to attach and survive initially, numbers dwindled until none were visible. We are therefore as of yet unable to establish a primary cancer cell model. Potential variables which could be of importance in future attempts to culture primary cells, include using a suitable attachment matrix, as well as optimizing the culture medium. Concerning PP2A in hypoxia in standard cell culture: MDA-MB-231 cells were cultured in a gas mixture of 5%CO2, 0.5%O2 for varying lengths of time, in growth medium containing only 1% FBS, where after cells were harvested and the following primary end-points investigated: (1.) The expression, phosphorylation and methylation of the catalytic subunit of PP2A; (2.) PP2A activity; and (3.) cell viability. Results: Following 72 hours hypoxia, Western blotting showed a decrease in total PP2A (control: 1.00±0.1426 arbitrary units (AU) vs hypoxia: 0.3513±0.07558 AU; n=3; p≤0.05), as well as unexpectedly a decrease in the relative phosphorylation of both PKB (phospho-tototal (p/t) ratio: control: 1.211±0.1820 AU vs hypoxia: 0.2088±0.03583 AU; n=3; p≤0.05) and ERK (control: 1.00±0.1519 AU vs hypoxia: 0.3493±0.05206 AU; n=3; p≤0.05). This led us to investigate shorter durations of hypoxia, specifically in the range between 2 and 8 hours. No significant changes in PP2A expression, post-translational modification or activity were observed, although there was a trend in increasing PP2A activity associated with hypoxia, which was highlighted by a significant increase in cells exposed to 6 hours of chemically stabilized HIF1ɑ (characteristic of hypoxia) (control: 134.7±33.74 AU vs positive control: 193.6±35.31 AU; n=2; p≤0.05). Four and six hours of hypoxia were consistently associated with a significant decrease in ATP. Discussion and Conclusion: Although we observed an interesting pattern of increased PP2A activity associated with hypoxia, our results failed to show a convincing link. Based on this, as well as literature, further research is needed: It would be interesting to investigate intermediate durations of less strenuous hypoxia (1% O2 instead of 0.5%). Modulating the activity of PP2A in hypoxia, followed by assessment of cell viability and pro-survival signalling would also contribute value.
Agtergrond: ‘n Tekort aan genoegsame bloedvoorsiening, met gepaardgaande hipoksie en ‘n tekort aan voedingstowwe, is kenmerkend van kankeragtige tumore. Die meganismes ter sprake by die aanpassing van kankerselle by hierdie kondisies is van belang as gevolg van moontlike kliniese toepaslikheid. Alhoewel onsterflike sellyne ekstensief gebruik is in kanker-navorsing, kan ‘n primêre kankersel-model moontlik bykomende inligting aangaande kankerbiologie oplewer. Proteïen fosfatase 2A (PP2A) is ‘n Serien/Threonien proteïen fosfatase wat vantevore betrek is by hipoksie, alhoewel teenstrydige inligting aangaande sy deelname gepubliseer is. Doelstellings: 1. Om ‘n primêre selkultuur model daar te stel deur selle te gebruik wat deur fynnaaldaspirasie (FNA) versamel is. 2. Om die effek van hipoksie op hierdie primêre selle te ondersoek. 3. Om die invloed van hipoksie op PP2A uitdrukking, post-sintese modifikasie en aktiwiteit in MDA-MB-231 selle te ondersoek. Ons hipotese was dat hipoksie ‘n afname in PP2A aktiwiteit tot gevolg sou hê om sodoende die fosforilering en aktivering van ensieme wat betrokke is by oorlewing en selvermeerdering te bevoordeel. Metodes: Nege FNA monsters is versamel by pasiënte in Tygerberg Hospitaal. Hierdie monsters is verwerk en in kultuur geplaas teen 37°C in gepaste groeimedium wat beide HEPES en epidermale groeifaktor bevat het. Selle is in kultuur onderhou vir tussen 1.5 en 6 weke. Medium is inisieel eers vervang na 3-4 dae en daarna elke tweede dag. Alhoewel dit eers voorgekom het of die selle geheg en oorleef het, het die getal selle uiteindelik afgeneem totdat niks sigaar was nie. Ons is dus tot op hede onsuksesvol in ons pogings om ‘n primêre kankersel-model daar te stel. Veranderlikes wat in aggeneem kan word in toekomstige pogings om primêre selle in kultuur te onderhou sluit in die gebruik van ‘n gepaste hegtings-matriks, asook optimalisering van die groeimedium. Aangaande die effek van hipoksie op PP2A in standaard selkultuur: MDA-MB-231 selle is onderhou in ‘n gasmengsel van 5%CO2, 0.5%O2 tesame met groemedium wat slegs 1% FBS bevat het vir wisselende tydsdure. Selle is daarna versamel en die volgende eindpunte ondersoek: (1.) Die uitdrukking, fosforilering en metilering van die katalitiese subeenheid van PP2A; (2.) PP2A aktiwiteit; en (3.) seloorlewing. Resultate: Western-kladanalise na 72 uur hipoksie het ‘n afname in totale PP2A (Kontrole: 1.00±0.1426 arbitrêre eenheide (AE) vs hipoksie: 0.3513±0.07558 AE; n=3; p≤0.05), asook ‘n onverwagse afname in die relatiewe fosforilering van beide PKB (fosfo-tot-totaal (p/t) verhouding: kontrole: 1.211±0.1820 AE vs hipoksie: 0.2088±0.03583 AE; n=3; p≤0.05) asook ERK (kontrole: 1.00±0.1519 AE vs hipoksie: 0.3493±0.05206 AE; n=3; p≤0.05) getoon. Hierdie resultate het ons genoop om korter periodes van hipoksie te ondersoek, spesifiek tussen 2 en 8 ure. Geen beduidende verskille in die uitdrukking, post-sintese modifikasie of aktiwiteit van PP2A is egter waargeneem nie, alhoewel PP2A aktiwiteit geneig het om toe te neem in assosiasie met hipoksie. Hierdie is uitgelig deur ‘n beduidende toename in PP2A aktiwiteit in selle blootgestel aan 6 uur van die chemiese stabilisering van HIF1ɑ (wat kenmerkend is van hipoksie) (kontrole: 134.7±33.74 AE vs positiewe kontrole: 193.6±35.31 AE; n=2; p≤0.05). Vier en ses uur van hipoksie was konstant geassosieer met ‘n afname in ATP. Bespreking en gevolgtrekking: Alhoewel ons ‘n interessante patroon van toenemende PP2A aktiwiteit in assosiasie met hipoksie waargeneem het, het ons resultate geen oortuigende verwantskappe getoon nie. In die lig hiervan, asook die literatuur, word verdere ondersoek vereis: Dit sal interessant wees om intermediêre tydsdure met minder ingrypende hipoksie (1% O2 instede van 0.5%) te ondersoek. Dit sal ook van waarde wees om die aktiwiteit van PP2A in hipoksie te moduleer, gevolg deur analises van sel-oorlewing en prooorlewings seintransduksie.
Description
Thesis (MSc)--Stellenbosch University, 2018.
Keywords
Needle aspiration biopsy, UCTD, Cell Culture, Breast -- Cancer, Hypoxia (Hematology)
Citation