resulting plasmid, pGLC, was checked by restriction analysis (figure 5.6). Digestion of pGLC with the enzyme Sac I generates bands of approximately 2.7, 0.5 and 6.2 kb whereas 0.5, 1.0 and 6kb fragments are generated from the original pBlueGB5'. pGL2 is linearized by digestion with Sac I due to the presence of a Sac I site in the MGS. The cloned 220bp PCR product was also sequenced to verify that it did not contain PCR- generated errors (not shown).
5.2.3.2 Construction of truncated bcl-2 promoter constructs (figure 5.7)
The parental plasmid pBlue GB5' was linearized with Not I, filled in with Klenow DNA polymerase and then digested with Xho I to release a Ikb fragment containing bcl-2 sequences, but lacking the most 5’ upstream sequences. Following digestion of the vector pGL2 with Sma I and Xho I, the Ikb Not l(blunt)-Xho I fragment was ligated into pGL2. Clones containing the correct insert were selected and digested with Xho I and Hind III. The PCR product described in section 5.2.3.1 (step 2) was also digested with Xho I and Hind III and then ligated into the modified pGL2 vector, generating the construct designated pGLE (figure 5.7). Constructs were verified by digesting with Sma I and AccI (figure 5.8). Digestion with these enzymes released a band of approximately 1.4kb from pGLE as expected, due to a Sma I/Acc I fragment in the bcl-2 sequence. An unresolved doublet of approximately 2.95 (AccI fragment) and 3.3 kb (Accl/Sma I fragment) was also obtained. The Sma I/Acc I digestion of pGLC yielded a Sma I band of 2kb. This is due to the presence of a 2kb Sma I fragment in the full length pGLC construct, which was removed from pGLE. An unresolved doublet of 2.85kb (Accl/Sma I fragment) and 2.95kb (AccI fragment, also in pGLE) is obtained when pGLC is digested with these enzymes in addition to the 1.4kb Sma I/Acc I fragment also found in pGLE.
5 2.3.3 Insertion of cytomegalovirus (CMV) immediate early promoter into pGL2 and pGLC
In order to obtain luciferase expression in HL60 cells, it was necessary to insert a powerful promoter into the reporter constructs. The plasmid pBK-CMV (Stratagene, UK) was digested with the restriction enzymes Nhe I and Nsi I to release a 600bp fragment containing the CMV promoter (figure 5.13). This fragment was gel purified and filled in with Klenow enzyme and with T4 DNA polymerase (section 2.1.10). The plasmid pGLC was digested with Not 1 (figure 5.13) and the blunt-ended CMV promoter ligated in. A clone containing the correct insert was selected and the resultant plasmid (designated pCMV-GLC) checked by digestion with Nco 1 (figure 5.14). The construct pGLC does not contain any Nco 1 sites. Therefore, digestion with Nco 1 would linearize pCMV-GLC and the original pGLC plasmid should remain uncut (figure 5.14). The plasmid pGL2 was cut with Sma 1, and the CMV promoter ligated in. Positive clones were identified by digestion with Nco 1 (figure 5.14). pGL2 does not contain a Nco 1 site. The CMV promotor insert contains a Nco 1 site at position 1531 (approximately centre of CMV). Nco 1 digestion therefore linearizes pCMV-GL2 and not pGL2.
5.2.4 Transfection of mammalian ceils
Initially 1 attempted to perform transfections using the established techniques of lipofection (Behr et a l, 1989; Loeffier et a l, 1990). In my hands, lipofection proved unsuccessful when applied to either HL60 promyelocytic or COS-7 cells. However, electroporation produced reliable results when optimized. A capacitance o f 960uF and a voltage of 300V at 25^C yielded the best results for both cell lines (figure 5.9). Luciferase activity was assayed 12 hours post electroporation.
5.2.5 Removal of 5' upstream regulatory elements abrogates bcl-2-driven luciferase activity
Ten micrograms of each of the constructs pGLC, pGL2 and pGLE were electroporated into subconfiuent COS-7 cells, as detailed in section 2.2.4. Extracts from these cells were assayed for luciferase activity 24 hours post transfection, normalized for protein content and expressed as relative luciferase units (figure 5.10). The promotorless pGL2 construct
yielded very low levels of luciferase activity. pGLC, containing the 4.5 kb bcl-2 5' untranslated region, reproducibly elicited a 10 fold increase in luciferase activity, demonstrating that the bcl-2 promoter elements could drive luciferase expression in COS- 7 cells. However, removal of 2kb of upstream sequences present in pGLC reduced the levels of luciferase expression by pGLE to basal levels, (mean ± sd; 10.9 ± 1.3 in cells transfected with pGLC versus 0.44 ± 0.06 in cells transfected with pGLE , n =3, p <0.01).
5.2.6 OKA does not downregulate bcl-2 promoter activity in COS cells
Treatment of COS-7 cells transfected with either the control plasmid pGL2 or the bcl-2- containing construct pGLC with OKA did not result in a significant alteration in the activity of the GLC relative to pGL2 (figure 5.11). In order to address the issue of whether a different apoptotic stimulus modulates bcl-2 promotor activity, the activity of the pGLC construct was measured following y irradiation. Bcl-2 mRNA is downregulated in response to 10 y irradiation in HL60 and in U937 cell lines (Chen et a l,
1995). A significant decrease in activity of the pGLC construct was observed relative to the empty pGL2 vector (figure 5.12), indicating that, in contrast to OKA, the sequences in the bcl-2 untranslated region are sensitive to downregulation following y irradiation in COS-7 cells (mean ± sd; 2.4 ± 0.36 in untreated cells versus 1.2 ± 0.2 in cells exposed to 10 Gy irradiation, n =3, p <0.05).
5.2.7 The bcl-2 negative regulatory element is functional in HL60 cells, but is not modulated by OKA
When the pGLC construct was tested in HL60 cells I was unable to obtain luciferase expression. The bcl-2 promoter is subject to highly potent downregulation by a NRE (discussed more fully in section 5.1.2). Removal of sections of this element still did not result in sufficient reproducible bcl-2 promoter-driven luciferase activity in HL60 cells (data not shown). I therefore constructed a heterologous promoter, (similar to that used by Young and Korsmeyer (1993) in their original studies on the NRE, by inserting the immediate early CMV promoter into the bcl-2 5' upstream untranslated sequences to test whether OKA acted on the bcl-2 NRE. Interestingly, when the activities of the constructs pCMYGL-2 and pCMVGLC were compared in HL60 cells, the bcl-2 sequences were found to exert a large inhibitory effect on transcription from the CMV promoter (figure
5.15). This is a probably as a consequence of the negative regulatory element as previously described by Young and Korsmeyer (1993) in REH and DHL-9 cell lines. Addition of OKA to HL60 cells transfected with pCMVGL-2 and pCMVGLC did not result in a significant alteration in luciferase expression from either plasmid (figure 5.15), indicating that this agent does not exert an effect on the negative regulatory element of bcl-2.
5.2.8 Bcl-2 expression is regulated post-transcriptionally by OKA in HL60 cells
Nuclear transcription run-off analysis was performed to determine whether bcl-2 gene transcription was controlled by other elements not contained in the constructs tested. However, although transcription of the actin gene was significantly repressed following OKA treatment, a decrease in transcription of bcl-2 mRNA was not observed following OKA treatment of HL60 cells. To verify that this experiment was actually assessing de novo RNA transcription per se, a-amanitin, an inhibitor of RNA polymerase, was incorporated into the transcription mix. The resulting radiolabelled RNA failed to hybridize to immobilized bcl-2 or actin probes (data not shown).
A 75-, CL O Q. < 48 72 9 6 0 2 4 C O S -7 HL60 Ti me ( hours)
Figure 5.1A. Induction of apoptosis in HL60 and in COS cells by OKA.
HL60 and COS-7 cells were treated with 20nM OKA. Cells were harvested at various times and cytospin preparations made. Apoptosis was quantified by the TdT-mediated dUTP Nick End Labelling (TUNEL) assay (section 2.6.3).
B
A ctin
Bcl-2 Bax
0
15 24 39 48 Hours Q 15 24 39 48Figure 5.1B Western blot analysis of HL60 cells treated with OKA.
HL60 protein extracts (25pg), prepared from cells treated for the indicated times with 20nM OKA were subjected to western blotting (section 2.3.2) and analyzed for expression o f bcl-2, bcl-X and bax proteins relative to (3 actin.