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PMX 500 – CCRF-CEM

PMX 500 – CCRF-CEM. XTT screening of compounds previously sent to Oncotest for screening against CCRF-CEM and a vincristine resistant subclone (CCRF-CEM/VCR) that overexpresses Pgp. Recent results comparable with Oncotest results. Concentration range to be adjusted.

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PMX 500 – CCRF-CEM

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  1. PMX 500 – CCRF-CEM XTT screening of compounds previously sent to Oncotest for screening against CCRF-CEM and a vincristine resistant subclone (CCRF-CEM/VCR) that overexpresses Pgp. Recent results comparable with Oncotest results. Concentration range to be adjusted. Vincristine resistant clones under development. Currently growing in 64 X IC50 (32 ng/ml). Plans to characterise resistant cells Western blot for Pgp expression. Can we recover sensitivity by co-treatment with Pgp inhibitor. e.g. Valspodar (PSC833) a cyclosporin A analogue.

  2. PMX 700 – SNB19V/VR/VR EPO cells Cells from Pharminox lab and EPO (at a passage number similar to those used to inoculate xenografts in current study) used in 7d MTT assay against temozolomide and compounds currently in SNB19VR xenograft study. SNB19VR and SNB19VR EPO cells grown without temozolomide for 5 consecutive weeks have been tested weekly for resistance to temozolomide. Each week represents 2 passages. No loss of resistance over 10 passages/35 days when cultured in vitro without Temozolomide.

  3. PMX 700 – Xenograft characterisation • MSH6 expressed in all U373 fragment samples. • Pattern for U373V and U373M fits with xenograft samples they are taken from (A3 and B2). • Did not receive U373VR sample fragments were taken from. MGMT (His tag) Mouse Ig LC MGMT U373 xenografts – EPO MV9750-01 – fragment inoculation Mouse Ig LC • Interference from mouse antibodies in blood trapped within samples. • Lower acrylamide concentration used in gels to try and resolve bands more. • Signal very high. Fatter bands. Results no clearer than before. MSH6 actin • M V VR VR M M V V • C2 C1 B3 B2 A3 A2 • U373 TC U373 xenografts • MV9750-01 fragments

  4. PMX 700 – Xenograft characterisation • This is the second run of these samples but MSH6 results are not consistent with previous run. • SNB19VR expressed MSH6 last time. Re-run samples. MGMT (His tag) Mouse Ig LC MGMT U373 xenografts – EPO MV9750 – cell/matrigel inoculation • MGMT blots not as good • Gel did not run evenly • Makes hard to interpret • Further bands from secondary antibody • Analysis of results is dependent on gel precision. • Use alternative antibody? • Rabbit derived –rabbit monoclonal antibody available Mouse Ig MSH6 actin • VR M V VR VR M M M V V V • C2 C1 B3 B2 B1 A3 A2 A1 • U373 TC U373 xenografts • MV9750 cells/matrigel

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