Costos de regulación

This work addressed the effect the state o f confluence o f the cells had on drug uptake, by employing both the confocal microscope and the flow cytometer. For flow cytometry, the difference in cellular drug uptake due to the effect o f confluence was observed in cells incubated with the drug in suspension and versus cells attached to the flasks. Stages o f cell confluence were described as sub- confluent (around 50% o f the flask covered with cells), confluent (around 90% of the flask covered with cells) and supra-confluent (cells were allowed to grow until the flask surface was completely covered and the cells became ‘heaped’ on top o f each other). All estimations o f the state o f confluence were made by eye and corroborated by an independent observer, skilled in culture techniques.

As shown by flow cytometry, when the cells were incubated with drug while in suspension in the same amount o f medium, the fewer cells present the more drug they took up. So the higher the number o f cells per volume o f medium the lower the fluorescence per cell; for example, M GH-Ul cells taking up MTZ when sub-confluent had a mean fluorescence o f 246 (arbitrary units) compared to confluent M GH-Ul cells which had a mean fluorescence o f 165 (arbitrary units). Both sub-confluent and confluent M G H -U 1 cells were incubated in suspension with MTZ in an identical way.

However, when the cells were incubated with drug still attached to each other and to a plastic surface, very confluent cultures took up more drug per cell than cells which were confluent and sub-confluent. In flow cytometry experiments, MTZ uptake by MCF-7/R cells, attached to the flask, produced very similar results regardless o f their stage o f confluence. The sensitive cells varied with their MTZ uptake depending on their state o f confluence, with the cells which were supraconfluent taking up twice as much drug as the confluent or sub-confluent cells at all MTZ concentrations.

Using the confocal microscope not only could the relative amount of intracellular drug, between cell lines, be viewed but also the intracellular localisation o f the drug investigated. There were differences with intracellular drug localisation within the same cell line depending on the confluence o f the cells. The

confluent and sub-confluent M GH-Ul cell line displayed a typical intracellular DOX distribution pattern with mainly nuclear drug localisation (Fig. 3.13C). However, the supra-confluent cells displayed a similar pattern o f DOX localisation to the resistant cells, with nuclear holes evident and rings o f bright perinuclear fluorescence (Fig. 3.13A). The confluent and sub-confluent resistant cells had similar patterns o f intracellular DOX localisation but the supra-confluent cells appeared have taken up more drug than the sub-confluent cells (Fig. 3.13B and D). The intracellular pattern o f IDA distribution did not change when comparing the supra-confluent and confluent / sub-confluent cells o f the M GH -U l and MGH- U l/R cell lines, respectively (Fig. 3.14). The supra-confluent and confluent / sub­ confluent sensitive cells had mainly perinuclear fluorescence with little nuclear IDA uptake. The amount o f fluorescence within the supra-confluent cells appeared to be more than within the confluent / sub-confluent, but no solid statement can be made as the cells in the supra-confluent cultures were very tightly packed together with little room for their cytoplasm to spread out.

The MCF-7 cells showed similar patterns o f IDA uptake but with the supra- confluent cells taking up much more drug than the confluent / sub-confluent cells (Fig. 3.15C and D). However, with MTZ there was a difference in intracellular localisation between supra-confluent and confluent / sub-confluent MCF-7 cells although both had taken up similar levels o f the drug (Fig. 3.15A and B). In both sets o f cells those on the edge o f the colony had taken up more MTZ than the rest. The supra-confluent MCF-7 cells displayed a pattern o f MTZ localisation often observed with drug uptake in tumour explants. Unlike sub-confluent cells, there was no definite cytoplasmic localisation o f MTZ in the supra-confluent cells. MTZ has not entered many o f these cells localising instead between the cells producing a ‘pavement effect’ o f fluorescence (Fig. 3.15A). This may also have occurred with the supra-confluent MCF-7 cells and IDA (Fig. 3.15C), however as the IDA fluorescence was so bright it was not possible to say for certain and the IDA may be intracellular. Different patterns o f drug uptake by supra-confluent and confluent / sub-confluent MCF-7 cells was also observed with DOX (Fig. 3.16). Supra- confluent cells show nuclear holes, with the majority o f DOX located in the cytoplasm producing intense fluorescence (Fig. 3.16A). In contrast, confluent and sub-confluent MCF-7 cells had low cytoplasmic fluorescence with DOX located

mainly in the nuclei especially within the nucleoli and the nuclear membranes (Fig. 3.16B).

In the majority o f these experiments there appeared to be increased drug uptake by supra-confluent cells compared to either the confluent or sub-confluent cells, with the intracellular drug distribution remaining unchanged. A change in the pattern o f intracellular drug distribution in supra-confluent and confluent / sub­ confluent cells was clearly shown, for example, by MCF-7 cells and DOX.

So in summary, different intracellular patterns o f drug uptake were observed supra-confluent cells compared to confluent / sub-confluent. Also, there generally appeared to be more drug taken up by supra-confluent cultures versus confluent / sub-confluent. These experiments led to the decision that all further work be performed on adherent cells when they had grown to a similar stage o f confluence judged, by eye, to be around 90% but definitely not supra-confluent. Cultures at different stages o f confluence have different percentages o f their cell populations at stages in the cell cycle, e.g. supra-confluent cultures will have a higher percentage o f cells in Go o f the cell cycle than confluent and sub-confluent cells the majority o f which are still dividing. Therefore, the effect that cell cycle may have on cellular drug uptake needs to be taken into account.

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Fig. 3.13. Intracellular DOX (10|ig/ml) localisation in confluent / sub- confluent compared to supra-confluent MGH-Ul and MGH-Ul/R cells, detected using the confocal microscope.

A and C : MGH-Ul cells B and D : MGH-Ul/R cells A and B : supra-confluent C and D : confluent / sub

confluent Nuclear fluorescence can be observed in the confluent / sub-confluent sensitive cells (C).

Nuclear holes with perinuclear fluorescence can be seen in confluent / sub-confluent resistant cells and in both sensitive and resistant supra-confluent cells (A,B & D).

Fig. 3.14. Intracellular IDA (10fj.g/ml) localisation in confluent / sub- confluent compared to supra-confluent MGH-Ul and MGH-Ul/R cells, detected using the confocal microscope.

A and C : MGH-Ul cells B and D : MGH-Ul/R cells A and B : supra-confluent C and D : confluent / sub-

confluent

Nuclear holes with perinuclear fluorescence can be seen in both sensitive and resistant, confluent / sub-confluent and

supra-confluent cells. (X300)

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Fig. 3.15. Intracellular MTZ (lOjig/ml) and IDA (10|ig/ml) localisation in confluent / sub- confluent compared to supra-confluent MCF-7 cells detected using the confocal microscope.

A and B : MTZ C and D : IDA

A and C : supra-confluent B and D : confluent / sub­ confluent

Nuclear holes with perinuclear fluorescence can be seen in both sensitive and resistant supra-confluent and confluent /

sub-confluent cells for both drugs.

A pavement effect o f fluorescence can be observed in supra-confluent sensitive cells with MTZ (A).

Fig. 3.16. Intracellular DOX (10|ig/ml) localisation in confluent / sub- confluent compared to supra-confluent MCF-7 cells, detected using the confocal microscope.

A: supra-confluent B: confluent / sub-confluent