WSB1 Induction by Hypoxia Mimetic - Dissertation Example

?The study of WSB1 Induction by Hypoxia Mimetic Cellular metabolism largely depends on oxygen availability and hypoxia-inducible factor 1 (HIF-1) is the main regulator of oxygen homeostasis. It is a transcription factor that is highly conserved that has an essential role in systemic and cellular homeostatic responses to hypoxia. HIF-1 is transcription factor, heterodimeric in nature, composed of constitutively expressed HIF-1? and hypoxia-inducible HIF-1?. The two subunits dimerize under hypoxic conditions, leading to HIF-1 complex translocation to the nucleus to bind to hypoxia-response elements (HREs) and activate the expression of target genes such as WBS 1 among others implicated in cell growth, survival and angiogenesis. The HIF-1 pathway is critical for normal development and growth and is associated with cancer pathophysiology, ischemia and inflammation. Hence, there is distinct interest in determining whether the compounds such as DMSO and DFO hypoxia mimetic modulate the HIF-1 signaling pathway. The aim of this experiment was to determine if these two hypoxia mimetics could induce the expression of WBS1 gene. In the determination of this chemicals’ ability to stimulate the HIF-1 signaling pathway as well as WBS1 induction. HCTII-6 and RKO cell lines were cultured and grown in media (GIBCO-DMEM) to approximately 75percent confluence. The confluent cells were later treated with this two chemical; DFO and DMSO. The cells were then harvested, quantified; proteins extracted then analyzed with western blotting. The western blot analysis indicated that these two chemicals induced the expression of WBS1 gene. According to the bands, for instance; the bands for DFO were brighter than those of DMOG probably it seems, DFO has more impact on the induction of the WBS1 gene than the DMOG. Literature review Oxygen (O2) levels have a very important role in governing several cellular pathways critical for survival of mammalian cell. By definition, Hypoxia is a reduction of tissue oxygen tension normal level. In hypoxia response, hypoxia-inducible factor 1 (HIF-1) in a mammalian cells is activated. It then regulates the transcription of genes associated with glycolysis, erythropoiesis, iron metabolism, cell survival and angiogenesis (Mole and Ratcliffe, 2008; Semenza, 2001). HIF-1 consists of two subunits: hypoxic responsive HIF-1? as well as constitutively expressed HIF-1?, also referred to as the aryl hydrocarbon receptor nuclear translocator (Wang and Semenza, 1995). There is rapid degradation of HIF-1? by ubiquitin-proteasome pathway under usual oxidation conditions, (Huang et al., 1998; Salceda and Caro, 1997). However, intracellular HIF-1? is stabilized under hypoxic conditions because of prolyl hydroxylase activity attenuation (Ivan et al., 2001; Jaakkola et al., 2001) which is needed in the initiation of proteasomal degradation (Stolze et al., 2006). The HIF-1? that is accumulated, heterodimerizes with HIF-1? then later translocates into the nucleus. It is known that the hypoxia-mimetic agents, DMOG and desferrioxamine (DFO), an iron chelator, induce hypoxia artificially by blocking HIF-1? degradation ( Hirsilaet al,2005).The complex of HIF-1 binds to DNA regulatory sequences referred to as hypoxia-response elements (HREs), that is located in the enhancer or promoter regions of HIF-1 target genes (Wenger et al., 2005). The expression of target gene is activated following the recruitment of transcriptional coactivators such as p300 and the cAMP response element–binding protein (CBP) (Lando et al., 2002),. Atleast seventy hypoxia target genes have been identified to date, (Wenger et al., 2005).This includesWBS1 genes. Discussion The hypothesis supporting this work is that the two Hypoxia mimetics induced the expression of WSB1 gene. With time, after addition of these two drugs the bands of HIF and WSB1 proteins became more visible. It is known that the hypoxia-mimetic agents, DMOG and desferrioxamine (DFO), an iron chelator, induce hypoxia artificially by blocking HIF-1? degradation( Hirsilaet al,2005).Therefore if this happens then it means that a lot of it will accumulate and may further result to the induction of WSB1 gene to be over expressed. Hence the bands looked brighter. The effect of the drugs is directly proportional to the HIF and WSB1 amount. According to the bands, for instance in figure 3, the bands for DFO are brighter than those of DMOG probably it could seem to be that DFO have more impact on the induction of the WBS1 gene than the DMOG. Also another reason could be that DMOG rate of action is slower than the DFO, may be if more time of reaction could have been given then this point could have been clarified. The impact of hypoxia-mimetic agents is similar to those following reduced levels of atmospheric oxygen (Jung and Kim, 2004) The membrane blot (in figure 1) probed with antibodies WBS1 and samples made from RKO cell line, had bands which were not brighter as compared to those shown in the membrane blot shown on (figure 2) which was probed with WSB2 antibody with samples from PRK cell line. The blot showed quite strong WSB2 bands at approximately 45-50 MW kDa. The whole of this was done as a test experiment. From this, it was clearly evident that wSB2 antibodies were the ones preferred to WBS1 but it was unfortunate there was no further experiment done on it because of time. When the blots were probed with Hif-1? antibody the bands were fairly good. The membranes were later probed with actin after getting blot images, According to the results the bands of actin were uniform indicating that there was consistency in the amount of samples loaded. The membranes blots shown in figure 3 and 4 had samples made from RKO and HCTC11-6 cell lines respectively .This was1st set of samples. As mentioned above, the blot in (figure 3) shows bright WSB1 bands at around 50 MW kDa .It is observed that those bands treated with DFO are slightly brighter than the bands shown on samples treated with DMOG. Furthermore the bands shown on samples from PRK cell line treated with DFO at 24 hrs were slightly brighter than at 6hrs and 0 hrs. This clearly shows that the impact of DFO is directly proportional to the amount of the HIF and WBS1 expression. This was not for the case with DMOG whereby the bands were almost similar at 6hrs and 24 hrs, this might be due to the slow action of this DMOG(less time range was given) or because of it has less effect on the induction of this WSB1 gene. For HCTC11-6 cell line, the bands were less bright than that in PRK cell line for DFO, but it was slightly brighter for the DMOG, the reason could be due to the PRK cell line supports the function of DFO much better than as the HCTC11-6 cell line could do. According to the results the bands of actin were uniform indicating that there was consistency in the amount of samples loaded. The bands in figure 5, 6 and 7 never came out quite well as expected despite the use of same samples as the one used in the membrane blots in figure 3 and 4 which had bands that were very good as expected. For instance, in figure 5 and 6, there was bright Hif-1 ? bands on samples treated with DFO but no bands on samples treated with DMOG was observed. On the other hands the WSB1 bands was hardly observed except there was one band observed after 24 hours on samples treated with DMOG. The membrane was also probed with actin which showed that the samples were loaded equally. The membrane blot (figure 6) shows Hif1-? band at both samples treated with DFO and DMOG. However, the bands on DFO are brighter which is similar to that of in figure 3 and 4 but not very good. On the other hand, WSB1 bands were only observed on samples treated with DFO for 6 hours. Probably this was not as expected and the reasons to this have been explored as below. Also the actin reprobe were not uniform for DMOG at 6hrs this indicated that the samples were slightly under loaded or may be due to reasons as below. The inconsistency in the results observed includes: no bands observed, faint bands (Weak Signal), Patchy uneven spots everywhere on the blot, too many bands on a blots, this might have resulted because of the following: No bands were observed. For instance in figure 5, 6, 7 and 8 bands were expected to be observed but were hardly seen, this could be due to: Insufficient antibody; may be the antibodies had low affinity to the protein of interest. To solve this, it is recommended to increase the concentration of the antibody (2-4 fold greater than standard starting concentration). (Haugland and You, 1998).The activity loss by the antibody could result to the problem. This could be confirmed through a Dot Blot. Also insufficient protein can result to this. It can be corrected by increasing the total protein amount loaded on gel. And a positive control is supposed to be used such as recombinant protein. The problem of no band could be due to Poor transfer of the proteins. This problem can be solved by wetting nitrocellulose membrane in transfer buffer or PVDF membrane in methanol before use. During transfer the contact between the membrane and gel should be perfect. (Lissilour and Godinot, 1990).Incomplete transfer can result to no bands as well. This can be corrected by optimizing the transfer time. This high MW protein required more time for transfer. To ensure complete transfer, the membrane was to be stained with Ponceau S, India Ink or Amido Black. prestained MW marker can be used to help optimize transfer.Over transfer can result to no band as well. This can be corrected by reducing the voltage or transfer time for proteins of low molecular weight (< 10 kDa). Protein Isoelectric point of interest which is >9 can also have an effect. An alternative buffer system with greater pH like CAPS (pH 10.5) ought to be used. (Amersham Pharmacia Biotech, 1998). Sodium Azide contamination can affect the band formation. To correct this, ensure that the buffers don’t have Sodium Azide because it can quench HRP signal. (Lydan and O’Day,1991).Insufficient time of incubation with primary antibody can affect the bands formation. The incubation time need to be extended to overnight at 4°C. (Schutzer et al, 1990) Faint bands observed in most of the blots could be due to: Low binding of protein-antibody.the number of washes needs to be reduced to minimum. NaCl concentration need to be reduced in Blotting Buffer for wash steps (standard range need to be 0.15M - 0.5M).Also NaCl concentration need to be reduced in Antibody Solution (standard range should be 0.15M - 0.5M).Insufficient antibody used can cause faint bands. This can be through low affinity of the antibody to protein of interest. Antibody concentration need to be increased (2-4 fold more than the standard starting concentration). (Hoffman and Jump, 1989). Faint bands can as well be as a result of insufficient protein, the total amount of protein loaded on gel should be increased to correct this problem. (Preac-Mursic etal, 1996). Some antigens may be masked by non-fat dry milk. Milk percentage in Antibody and Block Solutions need to be decreased or substitute with BSA (3%). Patchy uneven spots were observed all over in most of the blot, this could have been caused by: Reagents contamination can result to this spots. The buffers need to be checked for bacterial or particulate contaminate. Fresh reagents need to be prepared as well. Inadequate solution during washing or incubation might have resulted to this problem. To avoid this problem, the membrane must be immersed fully during antibody incubations and washing. (Haupl et al, 1993). Trapped air bubble in the membrane can result to this spots. Any air bubbles seen should be removed gently. During incubations, uneven agitation can result to this spots. Uniform agitation can be ensured through use of a rocker/shaker. Contaminated equipment can lead to this spots observed. The electrophoresis unit needs to be properly washed. Pieces of gel or protein remaining on the unit can get stuck to the membrane. The membrane should be washed thoroughly. Aggregation of HRP. HRP aggregates can be removed through filtering of the conjugate. Long exposure can result to this spots as well. Exposure time need to be reduced to correct this problem . Too many bands were observed on some of the blots as shown in figure 1, 2,3,4,5 and 6.This could have been due to: May be the antibody used was not specific for the protein. To correct this, need to reduce on the concentration of primary antibody. The total protein amount loaded on gel need to be reduced as well. Antigen affinity purified or mono-specific antibodies can be used to get only the required bands. (Linscott, 1999 ). Proteolytic antigen breakdown could have resulted to this problem. This is common, mostly if samples had been stored for a very long time or if the membranes or proteins are fractionated after tissue or cell homogenization. All additional bands observed in the blot are of lower molecular mass as compared to the full-length protein of interest. In order to prevent this, protease inhibitors could have been added. (Amersham Life Science, 2003).Too sensitive detection system or too much protein in every lane can lead to having many bands as expected. Gel overload is one of the most probable reasons for "ghost bands". Proteins immobilized might provide a concentrated surface that is adsorptive to which a given IgG might nonspecifically bind. This nonspecific binding, similarly might be uncovered when too sensitive detection systems like enhanced chemoluminescence are used. Usually a series of dilution of the starting material clarifies the signals that are artefactual. (Dattwyler et al, 1988).Inefficient blocking can as well lead to so many bands. There are several blocking agents described in the literature that includes; nonionic proteins and detergents. Blocking conditions alteration might solve the problem.(Salinovich and Montelaro,1986). Too low antigen concentration may lead to many bands. The SDS-PAGE resolution is limited. If the relative antigen concentration is too low (< 0.2% of total protein), it might be quite hard to detect. Enhancement of signal may then result to artificial bands appearance. Antigen enrichment by immune-precipitation or by fractionation needs to be considered.(Luft et al,1996). Secondary antibody non-specific binding can also lead to so many bands. A control need to be run with the secondary antibody alone (omit primary antibody). An alternative Secondary Antibody can be chosen if bands develop. Antigen affinity purified antibodies or mono-specific antibody need to be used here. (Dressler et al,1993) Conclusion The HIF-1 pathway is critical to normal development and growth and is associated with cancer pathophysiology, ischemia and inflammation. Hence, there is distinct interest in determining whether the compounds such as DMSO and DFO hypoxia mimetic modulate the HIF-1 signaling pathway. These results demonstrated the effectiveness of HCTII-6 and RKO cell lines cultured and grown in media (GIBCO-DMEM) in combination with secondary assays which was western blot for identification and determination of HIF-1? and WBS1 inducers and for distinguishing between these two inducers depending on their activation pattern of these hypoxic target genes. Identification and determination of environmental chemicals with HIF-1? and WBS1 activation activity in cell-based assays might be relevant for chemicals prioritization for future testing as hypoxia-response inducers in vivo. Bibliography Hirsila M, Koivunen P, Xu L, Seeley T, Kivirikko KI, Myllyharju J(2005). Effect of desferrioxamine and metals on the hydroxylases in the oxygen sensing pathway. FASEB J ,19:1308-10. Jung JY, Kim WJ. (2004). Involvement of mitochondrial- and Fas-mediated dual mechanism in CoCl2-induced apoptosis of rat PC12 cells. Neurosci Lett. 23:85-90. Amersham Life Science. (2003) n.d. A Guide to Membrane Blocking Conditions with ECL Western Blotting. Tech Tip 136. Amersham Life Science Inc., Arlington Heights, IL. Amersham Pharmacia Biotech.(1998). ECL Western Blotting Analysis System. Amersham Pharmacia Biotech, Piscataway, NJ. Haugland, R. P., and You, W. W. (1998). Coupling of antibodies with biotin. Meth. Mol. Biol. 80:173–183. Hoffman, W. L., and Jump, A. A. (1989). Inhibition of the streptavidin-biotin inter- action by milk. Anal. Biochem.181:318–320. Linscott, W. (1999). Linscott’s Directory of Immunological and Biological Reagents 10th ed. Linscott, Mill Valley, CA. Lissilour, S., and Godinot, C. (1990). Influence of SDS and methanol on protein electrotransfer to Immobilon P membranes in semidry blot systems. Biotech. 9:397–398, 400–401. Lydan, M. A., and O’Day, D. H. (1991). Endogenous biotinylated proteins in Dic-tyostelium discoideum Biochem. Biophys. Res. Commun.174:990–994. Salinovich, O., and Montelaro, R. C. (1986). Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 156:341–347. Wenger RH, Stiehl DP, Camenisch G. (2005).Integration of oxygen signaling at the consensus HRE. Sci. STKE 2005, re12. Wang GL, Semenza GL. (1995).Purification and characterization of hypoxia-inducible factor 1. J. Biol. Chem.270:1230-1237. Stolze IP, Mole DR, Ratcliffe PJ. (2006). Regulation of HIF: Prolyl hydroxylases. Novartis Found. Symp. 272:15-25. discussion 25–36. Semenza GL. (2001).Hypoxia-inducible factor 1: Oxygen homeostasis and disease pathophysiology. Trends Mol. Med. 7:345-350. Salceda S, Caro J. (1997) Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J. Biol. Chem. 272:22642-22647. Mole DR, Ratcliffe PJ. (2008). Cellular oxygen sensing in health and disease. Pediatr. Nephrol. 23:681-694. Lando D, Peet DJ, Whelan DA, Gorman JJ, Whitelaw ML.(2002). Asparagine hydroxylation of HIF transactivation domain: A hypoxic switch. Science. 295:858-861. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ. (2001). Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468-472. Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM, Lane WS, Kaelin WG Jr. (2001).HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing. Science 292:464-468. Huang LE, Gu J, Schau M, Bunn HF. (1998) Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. U. S. A. 95:7987-7992. Dressler F, Whalen JA, Reinhardt BN, Steer e AC. (1993). Western blotting in the serodiagnosis of Lyme disease. J Infect Dis. 167:392-400. Dattwyler RJ, Volkman DJ, Luft BJ. (1988). Seronegative Lyme disease: dissociation of specific T- and B-lymphocyte responses to Borrelia burgdorferi . N Engl J Med. 319:1441-6. Luft BJ, Dattwyler RJ, Johnson RC. (1996). Azithromycin compared with amoxicillin in the treatment of erythema migrans. Ann Intern Med. 124:785-91. Haupl T, Hahn G, Rittig M. (1993).Persistence of Borrelia burgdorferi in ligamentous tissue from a patient with chronic Lyme borreliosis. Arth Rheum. 36:1621-6. Preac-Mursic V, Marget W, Busch U, Pleterski Rigler D, Hagl S. (1996) Kill kinetics of Borrelia burgdorferi and bacterial findings in relation to the treatment of Lyme borreliosis. Infection. 24:9-18. Schutzer SE, Coyle PK, Belman AL. (1990) Sequestration of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme disease. Lancet. 335:312-5 Read More