Dear Leila Abaza,

All cell lines cannot be considered as "normal" , because they are immortalized (so, there is a transformaion of their genome expression). If you want to work on "normal" cells, that is more difficult because you cannot have the same number of them in survival or long term culture, you have to isolated them from organisms. In human, some usefull ones are circulating cells as blood cells, and you can isolate monocytes or polymorphonuclear cells (PMC) by centrifugation gradient. Monocytes can survive in flask, but htey differentiate them in macrophages, and in parallele PMC only survive. Indeed HL60 are derived from human mononuclear blood cells.

You can also use cells isolated from differents organs, and in the same way in animals.

But you have to remember that the expression of genes can be different in vitro than in vivo when cells are in culture.

Best regards

Didier JAMBOU

Dear Leila Abaza,

All cell lines cannot be considered as "normal" , because they are immortalized (so, there is a transformaion of their genome expression). If you want to work on "normal" cells, that is more difficult because you cannot have the same number of them in survival or long term culture, you have to isolated them from organisms. In human, some usefull ones are circulating cells as blood cells, and you can isolate monocytes or polymorphonuclear cells (PMC) by centrifugation gradient. Monocytes can survive in flask, but htey differentiate them in macrophages, and in parallele PMC only survive. Indeed HL60 are derived from human mononuclear blood cells.

You can also use cells isolated from differents organs, and in the same way in animals.

But you have to remember that the expression of genes can be different in vitro than in vivo when cells are in culture.

Best regards

Didier JAMBOU

Thank you for your quick and constructive answer that means that you dominate well the field of cell culture. As I mentioned before I want to study the anti cancer effect of some plant extracts and it’s possible to me to have the two types of cell lines (HL60 and MCF7). I will begin first by some screening tests: cytotoxicity assay, apoptosis assay and may be after some differentiation assays using HL60 cells. If I will find interesting results in vitro may be I will move up to in vivo step

I think that you can also test your plant extract on differentiated HL60 if it's the cells that you possess at present.

At the beginning, the first important test is simply cytotoxicity; because if it's toxic, this fact is most often corellated with toxicity in vivo (by experience in vitro cytotoxicity test that I have performed for pharmaceutical companies).

Then , if not, you can test the activity (on proliferation and apoptosis in cell culture) and study also simply the cell cycle (flow cytometry); then studing more specific actions to know the mechanism of action.

Before, in vivo test (do you think animal models ?), you can test your product on more physiological cells , and if you have not more than HL60 and MCF7, on circulating blood cells isolated as described in a lot of papers ; using your own blood or of your colleagues (control subject), and after you can used animal model tests.

These ways could have been mine in the past.

Best regards

Didier JAMBOU

@ Dr. Jambou: Just have a query. While using Lymphocytes for plant extract experiments, we have to give PHA (Phyto as what we often call it) for starting their division. So any how its cultured for a brief time and is loosing its "non-immortal" property and controlled division for a while. So can we really take it as a normal model?

it is the study of nature of cancer, replication pattern, its type, causes, factors responsible for its growth, progression and metastasis, impact on health, diagnosis and treatment methods, challenges towards developing drugs using modern tools and techniques etc......

Dear Dr Das,

When you establish a cell line that immortalizes, most often, the cell originated from a human or animal tumor (i.e Hela cells), where genes are already dysregulated; sometimes you can obtain a cell line from normal cells in long-term culture,without special stimulation, and the mechanism remains obscur (in vitro event that provoke mutation on cell dividing ?). Regarding to the different steps of cancerisation; the phase of immortalization corresponds to an inhibition of apoptosis and/or stimulation of permanent division (ie by tyrosine kinases/ras pathway activation).

When you have initialy normal cell in culture, it's not because they divides that they are abnormal.

So, when spontaneous division in cell culture don't arrive (as lymphocyte), you stimulate i by products that mimic physiological mechanism causing their natural division in vivo (stimulation by degradated antigen coupled to CMH and presented by macrophages). PHA (phytohemaglutinin A) is one of them; overs, like PMA (phorbomyristate actetate), ionophore A23187 directly stimulate the membrane for cell activation and division. But the cells stay "normals" , even dividing (but it's right that everybody uses these in vitro stimulants, but I don't know if geneticists verified that only norma ways of cell division are stimulated in this case).

If there is some expert advices on this subject, it could be interressant.

Best regards

Didier JAMBOU

dear friend,

a basic book called "A Computer Scientist's Guide to Cell Biology" will be helpful for u ill send this book to your mail please read this

@Dr. Jambou:

Thats right.

The bottomline: the cells (HPBLs) do not loose any of their in vivo properties except their growth kinetics, when stimulated in vitro by a mitogen. So other experiments, which do not involve the growth kinetics study can be fully authenticated in this regard. On the other hand when subjected to any plant extract or organic compunds for anti-cancer screening, if the growth kinetics of these cells' is studied (whether through classical FPG techniques or Flow), they may show delay or no delay in generation time or blocks in peaks at stages, but "may not" give a "proper indication". This is just my opinion.:)

But this fact too can't be neglected that this is the only way to study the fact (at least to my accessability).

When subjected to real animal trials, how (say for example) the Lymphocytes, will behave to the drug, even if the drug is meant to be targeted for some other case? Because any damage caused in vitro (DSBs or specially SSBs) are considerably repaired, unlike in vivo, in case of non dividing lymphocytes. What actually will be the effect on the Progenitor Cells, is somewhat unpredictable (in vivo). And we are talking only about one kind of cell here.

Although if we shift from humans, even mouse bone marrow helps parallely in predicting the effect of the drug to some extent. But Sir, I somehow believe that we definitely need more normal cell models for anti- cancer research. Or may be they are already there and i am not accessable to that knowledge as yet.

And thats very true that your reply on my query has helped me knowing about these facts.

And I am still a PhD student.:)

Best Regards

Abhishek

I agree with you. But at present, in the prossess of fundamental research in cancer we have, to my mind, different possibilities:

- continue to develop new in vitro models , more and more sophisticated , trying to reproduce in vivo conditons; but what ensure us that the simple fact of cultivating, or put cell surviving in culture can be consider as "normal" (so, in this case we never could't have "normal" in vitro cell models),

- or after these classical in vitro studies, continue directly on in vivo tumor models in animals,

- or finally try to develop new in vitro models (but could they be also "normals") aiming the in vitro reconstitution of organs (ie tridimentional cultures; co-cultures of hematopietic cells with environmental bone marrow cells + osteoclats, and sutdy of their interactions); or more "physiological": isolated and perfused entire organs where you apply your drugs ??

With best regards

Didier J

@ Dr Tatjana Zogovic-Kapsalis,

Dear Colleague,

I have described three possibilities of way of research (not all possibility) , that have been used or will be in future, but with no judgement (because if I am interesting in cancer biology, I didn't perform these experiments, even having perform researches on cell culture during nine year, in other fields of research; but not belonging to any old school). So, perhaps my opinion could be not pertinent.

However, I think all "models" won't never be the reality, and we'll be able only to approximate this one. In this case, which one could be the best approximation, before "doing the great jump": give for the first time a treatment to a person after all these experiments (see our problem in France with "Mediator" that have been prescribed many years and is dangerous for patients, with a lobbying of pharmaceutical industries to maintain this treatment).

But in balance, using stem cells as source of new therapeutic way appears to me to be a great hope for patients, already used for organ repair, by specific differentiation.

OK for always more inventivity, but with also always in our mind ethic considerations. And what serious researcher would go backwards, even by facility ?

Regards

Didier JAMBOU

I agree with Dr. Jambou. No one among us wants to go backwards in research, unless its the case of studying previous references. :)

Pioneer research towards constructing 3 dimensional organs for drug screening indeed can mimic the effect in normal in vivo conditions to the greatest extent (of course its my great dream to work on Tissue engineering).

Of course stem cell therapy is greatly helpful.

In addition to this I would like to mention that using medicinal plants too can really extract some very useful and unique results. Many researchers round the globe are working in this regard.

Since my time of joining research in this field I have always dreamt that some one somewhere in the world comes up with a wonder plant which can trigger apoptosis in ONLY cancer cells. :)

I think combinational therapy of chemical along with natural products may lower the effect of toxicity of the synthetic drugs too!!

@ Dipon: Thats true.

At times if the cytotoxicity of a chemical (Organic or Inorganic) is noticeably high but found to be specifically anti-cancerous, is being given as a combination with a lower dose of even radiation (e.g., 0.5-1.0 Gy of X-Rays). Here, both the drug and radiation doses are given at far lesser than their corresponding lethal doses, and have shown to produce a synergestic effect too in certain cases. This has been done in our lab while screening for anti-cancer properties of series of organotin drugs; and has shown promising results.

@ abhishek

Yea, thats absolutely true. But I think even lower doses of X-ray should be avoided because it will have a chance to mutate the other cells leading to further problems.Therefore we should try as much as possible with the combination of drugs instead of rays.

Avoiding radiation and coming up with a substitute with no side effects at all is always dreamt about by cancer biologists working with anti-cancer drugs throughout the world. Although use of any kind of treatment (chemical, herbal or be radiation) is done only after determining its LD50/30 value (in animal models) or IC50 value (generally in case of in vitro growing cells). LD50/30 stands for the minimum concentration of the drug with which 50% of the total treated test animals are alive (or dead) in 30 days; similarly the IC50 value stands for the minimum concentration of drug in which 50% of the cells are alive (or dead) before the control flask reaches 99-100% confluency.

Although the use of radiation is targeted keeping one thing in mind that the dose is far below its LD50 value. In case of mice the most accepted value for LD50 is 5 Gy.

Your point is correct that there may be a chance that it may cause a mutation in other normal cells; but at such a lower dose (as I mentioned above, i.e. 0.5 -1.0 Gy) of radiation normal cells even if are getting DNA damage will retain the ability of self repair or in last resort cases, will go for APOPTOSIS (as they are normal cells remember!). And look at the better part.....we are getting an increased/synergistic effect when using radiation in combination with the concerned drug! And in these cases the chemical combination generally kills the cancer cells and spares most of the normal ones as considerably less no. of the later are present in the targeted area. The fact lies here that all these drugs work with maximum expected efficacy only when THE TUMOR IS LOCALISED or hasn't reached malignancy. Any ways, its sure that the drug which will kill a cancer cell, will also kill a normal cell in 85% cases (unless its a drug which is formulated of discovered only to effect the cancer cells). See mathematically, if one normal cell and a cancer cell both die in one hit, we should be happy that the later has died and will mourn less for the normal cell as the body is capable of producing the earlier any time need occurs! :)

Yea, thats true, but prolonged exposure of rays although below LD50/30 value may cause some adverse effect on the the normal cells too as the rays can't be specific for the cancer cells.In cancer patients generally the repair mechanism will be more in the cancer cells as they will be in a hurry to divide and thereby repairing themselves otherwise with damaged DNA their cell cycle will get stuck at the S phase. But doing those , I think we are provoking the normal cells to become cancerous too. Even drugs where we determine the IC50 value, and generally we target the proteins or the mechanism which is required byt the cells to survice or to replicate. Its true that the normal cells get hit too but the rate of division of cancer cells is more than the normal cell , thereby intaking more amount of the drugs in the cancer cells than the normal cells. leading to its apoptosis or cell death.Therefore the rate at which the cells will reach will many folds higher in case of cancer cells as compared to the normal cells.Toxicity will be there, because no mdeicine will work without having any side effect, but for that toxicity we are preferring the combination with natural products which will have the anticancer properties and at the same time bringing down the IC50 value of the drug to minimal.

Dear Colleagues,

To my mind, you are all two right. And I think it undelines the problem of all therapeutic that aims to destroy cancer cells and "less" normal ones. And it concerns radiation as drugs because often acting on divisions and cell cycle.

In mammarian tumors, you treat with epirubicine and 5 to 10 years after can be responsible of myelodysplasia conducting to leukemia.

It could be the occcasion to begin a debate on another ways of cancer therapy: re-differentaition of cancer in normal cell (as retinoic acid and promyelocytic cell leukemia; the oldest one). If researchers working on this way could give light on it.

Best regards

Didier JAMBOU

I wonder that how cancer cells are so much vivid and beautiful in their organization, workability and sustainability. In simple words, all that a normal cell has, a cancer cell already pertains, but many things in the vice-versa is not true. These differences are targeted by the anti-cancer drug squad since decades, but some way or the other have to switch down to earlier protocols due to adverse side effects. One recent development which I came across in this thread itself is about Dr. Chao's work. Its about the increased concentration of Calreticulin in the CD 47 capped cancer cells.

(I am unable to post the link for that paper)

Regarding radiation, as I said before, its true that it may not be a very good source, but till now traditional practices, include radiation in cancer therapy apart from knowing its side effects. The sooner the substitute is found the better will the chances be.

Dipon's point here is worth talking. I would also like to add one thing here that lot depends too on what and how the subject's body might bear the radiation dose. It may be more worse than what you had explained and may be less than what we expect.

I would like to share something here. While doing some radioprotection experiments involving plant extracts in HPBLs, whole blood from 7 donors were taken (not keeping this in mind that the Aim was to see the resistance from radiation of each individual among each other).

Finally in the end of the year one interpretation came (which I showed to my supervisor but was said as unimportant); here I found that 2 of the donors in their control slides had shown an aberration percentage of a mere 9%, 2 showed 12-12.5 %, and rest 3 showed 16-17.3%!!!

Still I tried to check for their family origins and habits and found a considerable difference.

One girl among the donors was born and brought up in the mining areas of Meghalaya (Domiasiat). Must be an unfamiliar name, but, lot of fuss is running over here in this place to stop U-238 mining. I was happy to see that her blood showed an 8.8%!

I am still afraid to tell my supervisor about this! :)

It was understood that the habits cause a lot of radioresistance!

There's too a request to Dr. Jambou to tell us more about the re-differenciation of cancer in normal cells.

I agree to both the above points. Its true that a human body depends a lot on the enviormental surroundings. As in case of sickle cell anemia it is seen that african people are more ressistant to it than any other people in the world, because they have adapted a speicific mecahnism which protects them from it.Similarly, the drugs that we use today also have a problem, like its resistance inside the patient's body.Drugs like 5FU which are been used as drugs for cancer when the patient reaches the 4th stage , as it has lots of side effects, but still the drug is shown to be effective in only 40% patients , the reason behind this is the resistance of the cells against this drug. Similarly cancer cells seems to get resistance to most of the drugs that are used till now .I think we should also study how to overcome this resistance increasing the drug's effectiveness from 40% to 80 %