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A lymphoma is a cancer of the lymph system, there are two types one being Non-Hodgkin's lymphoma (NHL) which will be looked at in this article. Non-Hodgkin's lymphoma cells are derived from abnormally dividing lymphocyte cells (white blood cells) most commonly B-lymphocytes. The B-lymphocytes divide before they mature so they cannot fight infections. This means they are now abnormaly dividing and useless at protecting the body. When they start collecting in lymph nodes they grow into tumors.
What is Chemoresistance? Chemoresistance is the resistance of cancer cells to agents which are usually effective at destroying them. This is a big downfall which limits there uses and drastically reduces treatment options. Chemotherapy has been the major cause of failure in clinical cancer chemotherapy (Nooter & Stoter, 1996). Certain NHL patients are resistant to particular Chemotherapeutic agents, their cancer cells evade the cytotoxic effects of the drugs. This evading of cytotoxic effects is called Multi-drug resistance (MDR). A sign of MDR is that the cancer cells are less sensitive to the drugs used in chemotherapy but also the large number of drugs used, these of which have no obvious targets just random effects.
MDR can arise from structural and or functional changes in cell which includes mutated drug receptors. The MDR factor usually found in the majority of cells is the expression of a 170-180kDa energy-dependant efflux pump called P-glycoprotein (P-gp). This has a major role in regulating the concentration of chemotherapeutic agents that goes into a cells cytoplasm. Certain agents for example veramipil (calcium channel blocker) reverse the effect of P-gp therefore get around chemoresistance in cancer cells.
Since the introduction of Cyclophosphamide, Hydroxy daunorubiun, Oncovin and Prednisone (CHOP) a chemotherapy regime used to treat NHL patients. Select Patients have shown resistance to it. With this in mind other drugs in the regime are used for example anthracycline, hydroxy daunorubicin hydrochloride which is a substrate for P-GP. Vital information on mechanisms of resistance are acquired from in vitro models, after they have been exposed to hight levels of drugs.
Chemoresistance has been detected in a range of human cancers .E.g leukemia and malignant lymphomas. This is due to them expressing a cellular mutation wich leads to MDR. NHL patients at first are sensitive to CHOP but the treatment fails when a resistance disease and regimen-related toxicity (RRT) develops, both of which are a major causes of death for the patient. RRT occurs when a high number of chemotheraputic agents are given so much so that they cause a toxic effect on vital organs.
When studied measurable amounts of P-gp expression have been found in histological types of NHL. Clinical studies show P-gp expression is positively correlated to drug sensitivity in NHL patients. Other reports suggest no correlation between the two. Consequently in vivo, results suggest that P-gp expression is not linked to chemoresistance of NHL. This must mean there are additional mechanisms such as over expression of the p53 mutant protein. In vitro studies support this idea and findings show the abnormal p53 gene provides increased abnormal p53 protein which in-turn increases chemoresistance in NHL patients. Mutant forms on p53 protein enhance P-gp promoter activity wild type p53 protein represses P-gp promoter.
It has been proved that mutant p53 did not activate either the multi-drug resistance-related protein, MRP1, promoter or the endogenous gene. In contrast, mutant p53 strongly up-regulated the MDR1 promoter and expression of the endogenous MDR1 gene (Chin, Ueda, Pastan, Gottesman,1992; Sampath et al.,2001)
The response of DNA in tumour cells when combined with chemotheraputic agents such as doxorubicin with a P-GP antagonist such as verapamil in cells which over express p53 mutant protein.
This study is to reverse the action of P-GP in vitro with veramipil to try and mimic chemoresistance seen in vivo in doxorubicin based regimins in patients with over expression of p53 mutant protein. This was done in the SCGE assay where DNA damage was measured by and increase in tail moments.
It has been shown that P53 is frequently mutated in burks lymphoma derived raji lymphoblastoid cell sub-lines (thymidine kinse proficient and tyhmidine deficient) these sub lines share a common origin
Cell culture, the two types of raji cells (+,-) were grown in RPMI medium (grows lymphocytes/leukocytes) it also contained FBS (high growth factors less ab`s) 10% for + and 15% for - also contained antibiotics, cancer cells from the intestine epithelium (carcinoma) were maintained in a medium agin with FBS and antibiotics. The cell medium was changed every 72hrs after washing with streile PBS the cells were seeded, cultured until 70-80% confluent (running together, making them into one) split 1:3 over 2-3 weeks caco-2 cells between pass 7+9 were used for P-gp detection as a positive control since these cells are known to have P-gp
Cell treatment, raji cells (tk+ and tk-) were treated in microcentrifuge tubes with fluorouracil (treats cancer inhibits TK) to confirm sensitivity/resistance after this they were centrifuged and 890 ul of the supernatant was removed the reminder was mixed well 10ul of this was placed in a microcentrfuge to determine cell viability. The left over 100ul was mixed with 1% LMP agarose and the mixture was distributed over two BDH slides. Raji cells + leucocytes which were isolated from the clinical samples were were treated for 2hrs with doxorubicin (used in cancer chemotherapy treatment) with/without veramipil afterwards the SCGE assay was carried out and slides were made (measures DNA damage)
Cell viablilty chck, viablity was cheaked using trypan blue exclusion. At room temp 10ul of cells + 10ul of 0.4% trypan blue incubated for 3-5min. Cells which excluded dye were estimated using haemocytometer under BLMs preps with 80%+ visablity are sutiable for usage in SCGE assays
How to do a (SCGE) assay
raji tk+ cells over express p53 mutant protein (so It cant really check what is synthesised so the messed up “dna” goes un-noticed soo the cancer grows also sensitive to 5-fu (treats cancer, esp TK -/+ there was a big difference in dna damage with control and 40um response of raji cell tk- is also shown.
The response of tk +/-, after treatment with v,d, v+d in fig 2
with ref to fig 2
- v has little effect
- d causes increased DNA damage
- v+d is even higher
reversed the action of P-GP so increased in the raji cells response to D
This response was further examined in NHL patients cells those of which over express P53 mutant protein (4 patients and 1 healthy donor control) treatment was given as follows v,d,v+d = fig 3
- healthy + patient = similar DNA damage
- v+healthy +patient = little or no effects
- d+patients = damage more than healthy
when comparing d treated healthy the patient samplkes showed less damage = resistance to this drug. With d+v all the samples had higher damage compared to d on its own. TK- = more sensitive to d than tk+ as there is little cross resistance between 5-fu + d
V inhibits p-gp activity, action on p53 mutant protein, most chemotheraputics damage DNA/inhibit DNA synthesis so the conc of the agent in cytotoxic/cytostatic ranges in the cell cytoplasm after going through the memberane is important for treatment of malignancy in lymphomas.
The effects of the drug may be possible by direct/indirect assesmnet using SCGE assay. Altered DNA damage was investigated in TK+/- cells which over expressed p53 mutant protein + resistant to chemotherapy
5-FU was made to resemble pyrimidine bases u+t 5-FU can act with RNA, unmetabolised 5-FU is harmless to humans + tumour cells soo needs to be converted to active form to cause damage. To convert 5-FU to its harmfull form the cells take it up and metabolise it with enzymes + pathways TK. Raji TK- = Tk deficient TK+ = proficient. TK- celsl are more resistant than TK+ after treating with 5-FU as seen in fig.1
One aim of the study was to examine MDR, cross resistance between 5-FU + d was investigated to examine MDR. There was no x-resis in 5-fu + in TK- cells since they were more sensitive to d than 5-FU. If response to 5-fu+d is similar then MDR can be investigated.
5fu Is another potent cancer drug although its not effective at treating human lymphomas because it isnt a substrate for P-GP. Its maninly used to treat colon cancer. MDR might occur in drugs without a similare mode of action e.g. TK- which is more sensitive to D
1o aim = overcome chemoresis in NHL patients which high P53 mutant protein usung chemosensitizer model, Veramipil
One of the proection modes of cancer cells is and increaed efflux of cytotoxic compds out of the cell as they have an inc expression of membrane tranport proting P-GP
Pastan, reported overex by 60% in reoccuring lymphomas. No diff between V and dex fir revrsing the action of P-GP however cardioto for dex is lower than v, v also increases the cardiotox for D
in fig 3, clinical samples, low dna damage with D after adding V damage increased, although dna damage for control cells was higher after adding p-gp antagonist it appeared no less than in clinical samples.
P-gp was found in the NHL cells with over expressing p53 mutant protein. Cytotoxic cmpds inc V are substrates for p-gp and block the extrusion of anthracyclines therefore restricing the exit of drugs from the cell. Resuts were for NHL patients who had over expression of P53 mutant protine that v might help the exit of d therefore overcomming chemoresis.