Protein Concentration and Cytotoxicity

The article you are working with is the one I read. So these are my findings and conclusions from this paper (for figures refer to paper).

http://www.landesbioscience.com/journals/cc/article/yamadaCC3-9.pdf

Rusticyanin (Rus):

1. Effect on p53:

A. Translocation of p53 from cytoplasm to mitochondria that leads to ROS production (figure 3D)
B. No effect on nuclear p53 fraction (figure 3D)
C. Overall slight reduction of cellular p53 (particularly cytosol and mitochondria) (figure 2D and 3D)

2. Effect on p21 and p27:

A. Increase in both p21 and p27 that lead to CDK2 inhibition

3. Overall data:

A. Induces apoptosis presumably through caspase-8 activation
B. Apoptosis induced only if cell cycle is not arrested and cell lines are p53 positive.
C. Rusticyanin presumably acts via protein-protein interactions since it’s never present in the nucleus (figure 3D) and, therefore, cannot be transcription factor.

4. Cytotoxicity:

A. Cons. 50µg/ml (figure 3B) shows lowest cytotoxicity (lower than either 25µg/ml or 100µg/ml). Considering that Rus affects both the amount of p53 and its translocation into mitochondria, I would say that at cons. of 50µg/ml overall amount of p53 is affected more than translocation process.

5. Questions that need to be answered:

A. J774 cell line is heterozygous for p53 (info from another paper). It does not undergo apoptosis, but rather G1 arrest. Is that because 50% p53 is not enough to induce apoptosis OR cell cycle arrest overrides the apoptosis? I am not sure what mutations make this cell line cancerous. Suggestion: test the effect on wt cell line – exactly what you are doing.

B. It is unclear if Rus affects degradation/stability of p21, p27, and p53 OR it indirectly affects their transcription. Suggestion: northern blot for expression levels.

C. In regards to your last question, MTT and NBT measure mitochondrial enzyme activity, though enzymes are different, amount of each is proportional to viable cell number (thus raw data should be directly proportional to each other). If you process your data to determine treatment effect (treated – control) and plot that, then you should get MTT treated < MTT control = negative data, NBT treated > NBT control = positive data thus your relationship should become inverse: cell death = more ROS.

P.S. If you want to look up particular examples of the TF feedback that I was talking about previously – look up negative/positive feedback loops. One example I just came across is p53 regulation: p53/mdm2.

http://p53.free.fr/p53_info/mdm_family.html

p53 also activates KFL4 that also causes p53 degradation and p21 activation at the same time. If Rus simply causes stabilization of KFL4 and p27 (prevents their degradation) that would explain given results.

If you need more help with that, let me know.

I would appreciate it, if you registered to this site and used it directly.

Good luck!

Thanks for the info, was pretty helpful. I have made my peace with the variations in NBT results respective to different concentrations. I think I have enough to give plausible speculations which is all I can really do.

In regards to your last question, MTT and NBT measure mitochondrial enzyme activity, though enzymes are different, amount of each is proportional to viable cell number (thus raw data should be directly proportional to each other). If you process your data to determine treatment effect (treated – control) and plot that, then you should get MTT treated < MTT control = negative data, NBT treated > NBT control = positive data thus your relationship should become inverse: cell death = more ROS.

I’m not quite sure I understand what you mean here. As I understand, MTT measures mitochondrial enzyme activity to reflect cell proliferation, yes, but I thought NBT was just compound that acts as an antioxidant where the assay is based on the fact that the reduction of it, has a colour change associated with it that can be measured.
So I would think that if there were high readings for NBT assay which would directly reflect high amounts of superoxides, then the MTT readings, even in raw, would be low because there would be less cells and so less enzyme activity and therefore low absorbances
You mentioned processing the MTT results, I don’t quite understand how you explained to process them. But I don’t see why processing them in anyway would change the outcome because he raw results are pretty straight forward in that higher absorbance will indicate higher enzyme activity hence higher cell growth.

Let’s go back first. Data that you plotted – what is it?
For MTT you should have raw data of OD690 and the OD540 values as well as blank (negative control). Is OD in the graph the difference between the two? Corrected for the blank?

For NBT test you what are those numbers? Are they delta A550 nm/minute?

Now, I was talking about MTT and NBT readings over time when I said they should be directly proportional. Basically, more cells = higher MTT reading = higher NBT reading… You get the idea (your readings at 48 hours – practically no cells and all readings close to 0). However, when you are talking about readings post treatment with different concentrations it could be different.

You need to understand what it is you are actually measuring:

MTT – measures viable cell numbers and cytotoxicity
NBT – measures rate of ROS production in viable cells

So if your substance is cytotoxic, then you should observe decrease in MTT readings with increase in concentration of the substance indicating reduced cell numbers. If it is not cytotoxic, you should expect no deviation from negative control.

If your substance is cytotoxic and its cytotoxicity is due to ROS generation (as was proposed in that paper), then and only then you would expect to see the inverse relationship between MTT and NBT readings: as the rate of ROS generation increases, more cells undergo apoptosis. If your substance is cytotoxicity is not related to the rate of ROS generation, no inverse relationship would be observed and rate of ROS generation would be directly proportional to cell numbers.

S phase activity test could indicate several things: 1) Cell cycle arrest prior to S phase (as indicated in the paper), 2) Cell cycle delay (due to DNA damage, for example), 3) Decrease in overall cell proliferation.

If your substance causes cell cycle arrest prior to S phase or apoptosis, then increased concentrations of it should cause decrease in S phase readings. However, if your substance causes cell cycle delay the concentration might not have any apparent relationship until the mechanism is discovered. S phase data from some other experiments show similar characteristics (that is no predictable relationship between concentrations and S phase results at any particular time).

Assuming that your answers to my previous questions about data are yes, then it seems to me that: 1) Rus is not cytotoxic to your cell line (unless cytotoxicity of ~16% is sufficient? as observed at cons. =20 – you might want to check on that); 2) Rus might inhibit ROS generation at cons. ~10; 3) Since increased ROS generation rate (by ~6%) in conjunction with decreased cell numbers (by ~2%) was observed only at cons. =50 and the % difference is low, I would be inclined to assume that there is no relationship between ROS generation rates and cell death in this case. (of course, I don’t know the expected rate increase in ROS production due to increase in cell numbers so I cannot be sure).

Let’s go back first. Data that you plotted – what is it?

ok for starters all of my data for the MTT and the NBT assays was absorbance/optical density readings taken by spectrophotometry

For MTT you should have raw data of OD690 and the OD540 values as well as blank (negative control). Is OD in the graph the difference between the two? Corrected for the blank?

For NBT test you what are those numbers? Are they delta A550 nm/minute?

I'm afraid you've lost me here. are you saying i needed to include substances that gave absorbances of 690 and 540 along with my control? (sorry if that sounds stupid, but i dunno what you mean). What i can tell you is that the none of these results have had any corrections whatsoever. My control was simply the cell line on its own, i havent used this to correct any results or anything like that. My assays were done on microtiter plates and when they were ready i loaded em in the spectrophotometer, and directly plotted the readings it spat out.

For NBT test you what are those numbers? Are they delta A550 nm/minute?

I dont know what you mean here but the NBT readings, like the MTT ones, are simply the direct absorbance readings the spectrophotometer gave from the cell/protein sample mixtures. no corrections/manipulations

If your substance is cytotoxic and its cytotoxicity is due to ROS generation (as was proposed in that paper), then and only then you would expect to see the inverse relationship between MTT and NBT readings: as the rate of ROS generation increases, more cells undergo apoptosis. If your substance is cytotoxicity is not related to the rate of ROS generation, no inverse relationship would be observed and rate of ROS generation would be directly proportional to cell numbers

i think im starting to understand what you mean here. your saying that NBT correlates with MTT because if cells are being killed than there wouldnt be many left to produce the ROS, conversely the more cells you have, the higher potential for ROS generation. But as i understand it, in order for this to happen the ROS generation could not be playing a role in the cytotoxicity. now that i think about this it makes alot of sense and reminds me of the former use of Aspartate transaminase as a biomarker for liver disease where it would be released into the blood (where it could be measured) during liver trauma and so was used for diagnosis of liver disease. But it was deceptive because after extensive trauma there would be few liver cells remaining to release the enzyme and so the patient would falsely seem to be improving.

about the peculiar cell cycle results. by your theories it cant be g1 arrest since higher concentrations are actually stimulating S phase. i dont see how it could be decrease in cell proliferation since higher S phase should mean more proliferation and the MTT results seem to reflect this. the theory of cell cycle delay sounds interesting, dunno if i have interpreted your explanation right, but are you implying that it could be causing random delays that are actually independant from the concentrations and they just happened to take place in the cells recieving the lower concentrations? i dont suppose it is possible to get a link to these other experiments you mentioned?

these theories are very interesting and have given me a different perspective to my interpretations that i think could be enough to speculate some plausable answers, if i have interpreted you correctly?

First, let’s start with the measurements.

When you use spec, you (or someone) have to program it. Your readings are done at some wavelength. When you do MTT test you should get 2 readings: one at 690nm and another at 540nm for each sample. You also supposed to include blank – reagents without cells. Your ‘working OD’ should be a result calculated from this raw data. However, some specs are programmed to carry out these calculations giving you the result with or without accompanying raw data. You might want to check on that with your mentor. Similar thing applies to NBT test OD should be measured twice for each sample (both at 550 nm): once at 30sec and once at 5min30sec (for example) and calculated ‘working OD' reflects rate of ROS generation, however, as I understand there are several procedures available for NBT, so the details could be different. I suggest you check on that also.
Let me know when you find out.

P.S. I will post relevant links tomorrow.

oh ok, sorry i didnt know what you were referring to. Yes my tests were done at certain wavelengths. 540nm for MTT and 590 for NBT. however i never did different wavelengths tests like you suggest for MTT, only one wavelength. Also i only carried out the reading once. I didnt use any kind of blank, but the machinary is pretty big n fancey so it could have done it. I'm not sure about the durations, but i will check these out with my mentor.

anyway, my time in the lab is over and i cannot do anymore tests so whatever my results are for NBT wrong or right im going to have to assume they represent what is going in the cells. So far im explaining the directly proportional relationship between the NBT and MTT results under the theory that the more viable cells there are, the greater the potential for ROS generation which will be reflected in the results only if ROS is not causing cytotoxicity.

These are standard protocols for MTT and NBT tests. Your lab seems to use some customized protocols, but I think overall understanding of these tests should be useful.

http://www.aniara.com/pdf/literature/ICT-MTTKit.pdf
http://www.amsbio.com/datasheets%5Csuppliers%5CTrevigen%5C7500-100-K.pdf

Also read this paper and look at figure 4. That is what I said about time and dose dependant response in regards to S phase delay.

http://www.ehponline.org/members/1995/103-3/mangipudy-full.html

sorry been a bit busy lately

thanks for the links. i never considered that the concentrations could be acting at different times as suggested in that third article. Although my results suggest that the higher concentrations may be acting earlier than the lower ones.

Anyway, i think ive gotten a pretty good grip on things. I cant thank you enough for all your help, its really opened up my approach on these subjects and i'll be sure to mention you or the website under my acknowledgements section. : )

You are welcome!