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Turning Drug Resistance On Its Head Therapeutic Opportunity

Drug resistance is a major cause of cancer drug failure.

However where we can turn this resistance on its head it may offer therapeutic
opportunities. With the intriguing prospect that the more the resistance the more effective
may be our therapeutic response.

Bacteria are globally emerging resisting all known antibiotics which have helped extend our
lives and allowed major surgery. Because governments have been slow to step in to protect
us where pharmaceutical companies have for a number of reasons generally scaled back
on research for new antibiotic categories, we may be exposed to these infections for a
number of years before new antibiotic categories are clinically available.

There are good indications that our methods can be extended to bacteria and major
modifications under consideration make this more likely. Since clinically accepted drugs can
be used this can be expected to become quickly and economically available to fill the
therapeutic gap. Therefore a short preliminary paper is here brought forward.

Drug Resistance as a Therapeutic Opportunity

Bernard M. Turner
SUMMARY
The wall of resistance to cancer and bacterial drugs may to some extent be an artefact of
prevailing chemotherapy. This comprehensive adaptive response to drugs is fortunately largely
selective to cancer and many infectious diseases. Here we show in vitro that this may give us an
opportunity for a corresponding on-going chemotherapy where the resistance to a drug is used
to set up the target cells to activate within the cells a subsequently delivered counterdrug.
This may be of comparable if not more selectivity and efficacy than the drug resisted and can
sometimes use clinically accepted drugs for a quicker and more economical development as
well as increase the cost-effectiveness of targeted drugs. Moreover, this may be modified to
set up conditions favouring the delay or even reversal of resistance to a drug by codosing it
with the counterdrug. This can be empirical in not necessarily requiring the resistance
mechanism to be known. The prevailing cancer chemotherapy is becoming increasingly
unsustainable and bacteria resistant to almost all known antibiotics are globally emerging, so
that this alternative strategy should be a timely challenge to consider clinically developing and
extending.

Major causes of cancer drug failure are drug resistance, toxicity limiting the dosage and metastases.
A dearth of major new drugs to replace those coming off patents despite a steady rise in R&D
expenditure1 has been attributed to a lack of sufficient innovation.2 This with genetic drug
competition has caught up with the pharmaceutical companies3 leading to downsizing, redundancies

and a scramble for M&A as well as efforts to reduce the enormous cost and time of drug
development.4
Antibiotics have an additional built-in obsolescence (Fig 1) discouraging R&D. For while cancer
drug resistance terminates with the death of a patient from whatever cause, bacterial drug resistance
can be cumulative, spreading rapidly horizontally in hospitals and other communities by clonal
expansion as well as plasmids between bacterial species, accelerated by global travel. This combined
with the misuse of antibiotics in humans and farm animals and the lack of leadership by governments
in regulating this and promoting R&D where pharmaceutical companies are lacking is a recipe for
the global incubation of bacteria now emerging. Resistant to almost all know antibiotics, e.g.
NDM-1-metallo-beta-lactamase plasmids, know for some thirteen years,5 now spreading globally in
pathogenic bacteria.
The completion of the Human Genomic Project has given a great impetus to the search for new
anticancer targets for Ehrlichs magic bullets.6 However, this is becoming increasingly
unsustainable with the Wests ageing population as currently emphasised.7 Cancer care and
treatment alone is predicted to bankrupt the UKs NHS by 20258 and is causing serious concern in
USA. There is an increasing clash between biomedical progress and equity yet to be confronted.9
It would therefore appear timely to pause and consider whether a reorientation of the prevailing trend
to complexity largely generic diagnostically and therapeutically is possible without sacrificing drug
efficacy. This article briefly outlines one such alternative approach with in vitro example of each as a
step towards the end objectives: 1) to overcome drug resistance, 2) use the latter as an opportunity
for delivering a more effective counterdrug, 3) where possible with clinically accepted drugs for
quicker and more economical development to the clinical stage equivalent to late-stage drug
development, 4) more sustainable to the Wests health services financial constraints and 5) more
accessible to the low-income economies where some 70% of cancer and most infectious diseases

occur. 6) Where possible an empirical method for simplicity, economy as well as speed of response
when used. 7) Adding these advantages to targeted drugs, including those selected by personalized
cancer treatment to make these more sustainable.8-9
Drug resistance as a selective target.
As well as disclosing many new potential drug targets genomic projects have also shown the
complexity of interacting networks and feed-back loops within10 and between11 the genomic,
transcriptional and translational levels and with epigenetics. Blocks one target with a silver [costly]
magic bullet and alternative pathways resist this restoring the initial or an alternative equilibrium.
Like squeezing a balloon.12
This robust13 adaptive response is perceived as a wall of resistance. Differing from organismal
evolution, neoplastic cells revert to asexual, unicellular organisms in expanding clonally. Evolving in
a time-scale of years and bacteria even of weeks instead of millennia. There are over 200 cancers
with clonal heterogeneity within each and of course many infectious diseases, with many
mechanisms of drug resistance. However, in this complexity we should not lose sight that drug
resistance is fortunately largely selective to cancers and infectious organisms and much rarer in
normal human tissues over shorter time spans and then quite likely epigenetic.
Since Nature has will but cannot see,14 drug resistance may therefore offer us a favourable
opportunity to set up the target cells to a simpler, wide-spectrum chemotherapy. Based more upon
this adaptive response than molecular complexity, for comparable if not more efficacy and
selectively than the drug resisted. As well as increasing the cost-effectiveness of complex targeted
cancer drugs allowing us to use where possible lower cost, clinically accepted drugs.
This turns drug resistance on its head in the sense that the more the resistance the more effective can
be the counterdrug. Depending upon a number of factors including the therapeutic gain (tg) of

counterdrug 2 (C.Dr 2) when it is activated by the resistance R1 to C.Dr 2* (Fig 2). Tg is here
defined as the LD50 ratio C.Dr 2*: C.Dr 2.
Overcoming drug resistance (Fig 2) Cancer.
A drug 1 (Dr 1) develops or is further induced to develop a resistance R1. This in turn sets up the
target cell to activate a subsequently delivered C.Dr 2 (prodrug). Since this is of lower toxicity it can
be dosed at higher concentrations, generating endogenously (or at the cell wall) a higher
concentration of activated C.Dr 2* than can usually be given and with substantially lower sideeffects.
Where the gene induced to express R1 is also widely distributed in evolution this may allow a wide,
therapeutic spectrum based upon the foregoing specificity. Cytidine deaminase (CD) is one such
gene in our prototype formulation (Fig 3). With homologies throughout evolution from archaic
bacteria to humans. As a family of metabolic and activation induced (AID) members15-18 which can
reach a high level of expression in ALL, AML and CML 19-20 and often other cancers when
activated. Deaminating to inactivate the long-established clinical drug 1-beta-D-arabinofuranosyl
cytosine (AraC) and activating the subsequently delivered, clinically well-established 5-fluocytosine
to 5-fluorouracil (5FU) (Fig 3). 5FU has only a narrow therapeutic window (TW) difficult to control
in vivo between effective dose and high toxicity. Where the CD expression developed or further
induced is high, this can generate endogenously a relatively high 5FU concentration equivalent to
high dose chemotherapy (HDC) with 5FU 21 but with much reduced side effects expected. Which
may approach a knock-out (KO) dose important to prevent time for further acquired resistance R3
such as thymine synthase (TS) resistance developing, A modified method of refs22-23 is also being
considered if required to reduce toxicity further and rescue normal host cells.

Alveolar lung adenocarcinoma established cell line A549 gave a favourable response in vitro (Fig 3),
The CD resistance assayed as24 increases steeply with multiple doses of 0.5mg/ml (net) AraC, which
has at this suboptimal dose a reduced toxicity.
Where a gene such as CD already exists in the human genome, gene transfection by injection into the
tumours25 for selectivity would be unnecessary and miss metastases. Thus the thymidine kinase gene
in a replication-deficient adenoviral carrier has been transfected into a highly malignant brain glioma
to sensitize it to gangliovar.26 Whereas this cancer and other primary and secondary brain tumours
would be potentially favourable candidates for the prototype formulation (Fig 3). Since AraC is
tolerated by the CNS,27 the brain has a particularly low background CD28 and analogues are under
consideration to reduce cerebral damage.
Bacteria
Serious problems of bacterial antibiotic resistance may also be by-passed by resorting to the
AraC/5FC formulations or analogues particularly if lower toxic modifications are successful or these
are incorporated into antibiotic formulations (below).
Streptococcal and faecal cells were made resistant to a serial dilution of AraC applied x1-2 daily
mostly in log growth, followed by 1-2 doses of 120 ug/ml 5FC (net) and assayed by a relative
turbidity (RT) method (Fig 5). Streptococcus showed a marked inverse relationship between RT and
5FC concentration. 5FC was also under these conditions bacteriocidal to faecal cells, increasing
linearly with a serial dilution of the AraC administered earlier to set up the cells.
Methicillin induces beta-lactamase overproducted29 in MRSA. This can spread to other bacterial
species by plasmids and secreted beta-lactamases in infected or absess fluids30. A remarkably wide
range of beta-lactamases can be induced in resistance31. Counterdrugs to this cephalosporin-3-R2
derivatives, corelease the substituent R2 upon beta-lactamase cleavage of the beta-lactam bond,

usefully also for beta-lactamase assays32. Considering this had a fairly low tg quite satisfactory in
vitro results were obtained (Fig 6).
Drug and counterdrug schedules.
A number were tested in vitro to approach stepwise a KO dose33 with cancer or bacterial cells. 1)
The resistance R1 (Fig 2) may be built up be a succession of doses of Dr 1 to activate one or a few
doses of C.Dr 2 (Figs 4-6). 2) The cell platform burden reduced stepwise by a succession of Dr 1 and
C.Dr 2 interspersed with other drugs (Fig 7). 3) Dr 1 and C.Dr 2 can reciprocally set up for each
other the raising or lowering respectively of R1 (Fig 2). Thus when AraC and 5Fc were closely
alternated for two successive cycles (Fig 3) over several hours under log growth conditions changing
the medium at each step, there was a stepwise reduction to 85% bacteriocidal (Fig 8). 4) Of
particular note formulations intended to delay drug resistance may reach towards a KO for reasons
suggested below. It will be interesting to see how far these schedules can be adapted to in vivo
conditions, such as Fig 8 as an effective alternative to HDC of 5FU21 by continuous intravenous
infusion with lower toxicity.34
Delaying drug resistance (Fig 9).
The success in codosing two or more HIV antiviral drugs has encouraged extension to anticancer
drugs with only limited success. However, where R1 and Dr 1 and tg of C.Dr 2 are sufficiently high
and the C.Dr 2 is codosed with a drug 3 (Dr3), resistance R2 to Dr 3 may be suppressed. For there
would be a steep rise in C.Dr 2* activity when cell activity resumes as R2 develops. Since this may
be close to if not at a KO (Fig 10), the target cells may have become trapped between Dr 1 and a
steeply rising C.Dr 2* activity (to be confirmed). Coupling a C.Dr2 with a Dr3 (Fig 9) may also
come to have the following advantages for cancer and bacterial applications:-

(i) Delays passive cancer or bacteria cells resuming activity and (with cancer) metastasising, (ii) This
can be empirical, not always requiring Dr 3 resistance R2 mechanism to be identified at least
initially. (iii) Which may be particularly useful where there are complex resistances e.g. to EGF-R or
PDGF-R inhibitors or (iv) a rebound of resistant cancer cells after a successful course of treatment or
(v) against a histologically confirmed cancer metastasis from cancers of unknown primary sites (a
substantial cause of cancer death). (vi) Where a rapid response by an antibacterial drug to an
infection is required. e.g. to B.anthracis, haemolytic strains of E.Coli such as 0104:H4, or an initially
unknown bacterial infection.
Reversing drug resistance.
Drug resistance can sometimes be induced (Fig 2) or reversed (Fig 11) across the perceived wall of
resistance. Reversed by varying the formulation delaying drug resistance (Fig 9). In either direction
selecting for minimizing the generation of high tg C.Dr 2* by drug resistance R1 or R2 respectively.
Thus the resistance of a Streptococcus to penicillin G was reversed by first incubating the bacteria
with suboptimal concentrations of penicillin G codosed with a full concentration if 5FC, followed by
assaying penicillin G activity with an optimal dose of penicillin G in fresh medium (Fig 12). The
reversion of resistance might expect to sometimes overrun weak original drug efficiency, increasing
this by selecting for e.g. increased drug influx, reduced efflux or even increased conformation of the
target to Dr 3. Increasing Dr 3 efficacy and widening its useful spectrum (to be confirmed).
Discussion.
The perceived wall of resistance and dose limits to cancer drugs may sometimes be overcome and
this extended to bacteria. Not only as a simpler alternative to prevailing methods but also to seek to
increase the efficacy of targeted cancer drugs. Where these are selected by personalized cancer
treatment it may help to increase sustainability which may come to limit application.8-9

For reasons given these methods may be timely to develop quite quickly and economically and
consider how far they may be adapted to clinical practice. For where we can turn drug resistance on
its head, the more the resistance the more effective may be the ongoing chemotherapy. Turning the
resistance we fear to advantage. Rapidly resisting organisms such as HIV and the flu retroviruses,
malignant melanomas and brain gliomas becoming candidates and antiangeniosis drugs35 reaching a
fuller application.

Acknowledgments: I thank Mrs P. J. Turner for unstinting and skilled collaboration; Mr J. M.

Turner for ongoing IT support; Mrs E. M. Kiddle for IT assistance; Mr R. Sharp and Mr A.
Williams, Gigstream plc, Oxford UK for computer research advice; Mrs J, Collard for assistance
with photomicrography and photography. Competing interests: Patent applications.

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15. Laliberte J & Momparler RL. Human cytidine deaminase purification of enzyme, cloning and
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18. Yang C. et al. Cloning and nucleotide sequence of the Escheria coli cytidine deaminase gene.
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19. HO DHW, Frei E.Clinical Pharmacology of 1-beta-D-arabinofuranosyl cytosine.
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20. Muller W. and Zahn RK. Pharmacology of Cytobarine. Cancer Res. 34, 1103-1107 (1979)
21. Nieboer P. et al. High dose 5-Fluorouracil Chemotherapy. Cancer Treatment Review, 31, 210225 (2005).
22. van Graenigen CJ, Peters GJ, Leyva A. et al. Reversal of 5-Fluorouracil Induced
myclosuppression by Prolonged Administration of High-Dose Uridine. J.Nat.Cancer Inst. 81(2),
157-162 (1989).
23. Leyva et al. Phase 1 & Pharmacokinetic studies of High-Dose Uridine intended for rescue from
5-Fluorouracil Toxicity Cancer Res. 44, 5928-5933 (1984).
24. Anderson L. et al. Cytidine deaminase essay Arch. Microbiol. 152, 115-118 (1989).
25. Harris JD, Guttierrez AA, Hurst HC, et al. Gene Therapy for Cancer Using Tumour-specific
prodrug activation. Gene Therapy 1, 1-6 (1994).

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26. Sandmair AM. et al. Thymidine kinase gene therapy for human glioma Using replication
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29. Keseru JS, Gal Z, Barabas G.et al. Investigation of beta-lactamases in Clinical Isolates of
Staphylococcus aureus for Further Explanation of Boderline Methicillin Resistance.
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12

LEGENDS.
Fig 1. Resistance to antibiotics develops quite rapidly. The beginning of the arrows when a given
antibiotic was clinically introduced, the tip of the arrow when the first clinical case of
resistance to it was reported.
Fig 2. Overcoming drug resistance. When resistance R1 to drug 1 (Dr 1) develops or is further
induced it activates a subsequently delivered lower toxic counterdrug 2 (prodrug) (C.Dr 2) to
a cytocidal (or apoptotic) counterdrug 2 (C.Dr 2*).
Fig 3. Prototype formulation of Fig 2. One or more doses of AraC (Dr 1) are given until resistance
R1 (cytidine deaminase) resistance develops. 5FC (C.Dr 2) is then added at relatively high
concentration where it becomes deaminated by R1 to cytocidal 5FU (C, Dr 2*).
Fig 4. CD resistance to AraC sets up (sensitizes) cancer cells to counterdrug 5FC (Fig 3). Alveolar
type 2 lung adenocarcinoma cell line A549 dosed daily for 7d with serial dilution of AraC
changing medium daily. On +8d one dose of 5FC in fresh medium added. Residual cells
assayed as described in Methods. 1,0125. 3,025. 4,05 g/ml (net) AraC. 2,cells, medium
only. 5, resistance overcome by 5FC.
Fig 5. Overcoming bacterial resistance to AraC with counterdrug 5FC (Fig 3).
A serial dilution of AraC was added daily for four days with reinoculation into fresh medium
daily. 1,faecal. 3,a Streptococcus. Counterdrug 5FC was then added to 2,faecal and
4,Streptococcous and assayed by relative turbidity~19h later. Resistance overcome by the
5FC to 5,faecal and 6,Streptococcus. All concentrations within clinically accepted limits.
Fig 6. Overcoming penicillin resistance (Drug 1) with a counterdrug 2 (Fig 2). A penicillin-G
resistant Streptococcus was incubated with a beta-lactamase counterdrug and assayed 19h
13

later. 3,penicillin-G sensitive, 1-resistant bacteria. 5, penicillin resistant cells are sensitive
to counterdrug, but 4,penicillin sensitive bacteria are not.
Fig 7. Discrete, short multiple step dosing reduces the cell burden towards zero. A Streptococcus
inoculated successively into fresh medium each time in 25cm2 cell culture flasks and
incubated 36-37C.(a)-(a*) AraC, (c)-(c) penicillin G, (d)-(d) 5FC, (e)-(e) AZT. Penicillin
G resistance 1> 2>3. 1,High AraC, low 5F activity, 2, very high AraC, fairly low 5FC
activity. 3,AraC and 5FC fairly low activity. All high AZT activity. Assayed relative turbidity
as inverse of time to reach a standard turbidity. Clinically accepted concentrations.
Fig 8. Discrete rapid alternation of drug 1 and counterdrug 2 (Fig 2) reduces cell load towards zero.
A Streptococcus model. Innoculi successively into fresh medium in 18-well plates incubated
at 36-37C for 2 or 3h alternatively with 2,5FC and 1,AraC in two cycles Fig 3. Relative
turbidity as for Fig 7.
Fig 9. Delaying drug resistance.
A. Dr 1* is codosed with its counterdrug C.Dr 2. Deactivating Dr 1* to Dr 1 by R1 is delayed
because the cancer or bacterial cells renewed activity activates the C.Dr 2 of high therapeutic
gain (as defined) to C.Dr 2*.
B. Codosing C.Dr 2 with a drug 3 can also for the same reasons delay the resistance R3
deactivating another drug Dr 3* becoming deactivated to Dr 3 by another resistance R3.
Fig 10. Delaying cancer drug resistance. Alveolar type 2 human lung adenocarcinoma cell line A549
incubated with 5 successive doses over nine days with change of media each time. 3,AraC,
challenged at +39d with 5FC. 4, 5FC. 1,AraC + 5FC. 7,cells in medium only. All
concentrations within clinically accepted concentrations. The absence of recovery of 1 after
44d+ in fresh medium no agents indicated an approach to a KO dose.
14

Fig 11. Reversal of bacterial or cancer drug resistance. Fig 9B can be reversed when the cells are
resistant to a Dr 3. A suboptimal concentration 1/n x Dr 3 is codosed with an optimal
concentration of C. Dr 2. Dr 3 can develop activity to Dr 3* to inhibit the cell resuming
activity and activating the high therapeutic gain C. Dr 2 to C. Dr 2*. Such that an optimal
dose of Dr 3 alone (or other combination) subsequently delivered is cytocidal.
Fig 12. Reversal of penicillin-G resistance (Fig 11). 1, A Streptococcus presensitized to activate 5FC
(Fig 3) was incubated with a serial suboptimal concentration of penicillin-G. codosed with a
full dose of 5FC and then assayed with an optimal dose of penicillin-G. 2,as 1 but the bacteria
not presensitized to 5FC. a~70% reversion of penicillin activity, b before resistance
developed. 3, As 1 but not presensitized and no 5FC added.

15

METHODS.
Materials. Sigma-Aldrich (Poole, Dorset, UK), Calbiochem (Merek Bio-Sciences Ltd, Beeston,
Notts, UK), Molecular Probes Europe BV (Leiden, The Netherlands), Upstate Ltd (Milton Keynes,
UK).
Microscopy. Tungsten (Tenstile,Prior), Mercury (intense UV lamp) with filters. X1-x100
transmission (Koeller), incident, phase contrast. Microdensitometer (Evans Electroselenium, UK) or
35mm camera (Leica/Leitz) in vertical tube of a binocular microscope. UV/visible Unicam
spectrophotometer. The latter and microscope stage with temperature control cell holders and
chambers respectively, circulated by water from Haake F3 Digital water bath (Seven Sales, Bristol,
UK).
Cancer cell culture and procedures. The cells were incubated at 37C in treated 75cm2 or 25cm2
cell culture flasks or 6-96 well-plates with sealed lids (Corning Inc. Corning, NY, USA) in media
recommended by cell suppliers, usually with 10% foetal bovine serum supplemented with Lglutamate, penicillin G, streptomycin sulphate and sometimes amphotericin B. Cells trypinised
(0.25% tyrpsin (1,250) / 0.02% EDTA) to detach. PC-3 cells also had 1% neaas and 0.01%
collagenase or no collagenase for monolayer or soft agar (for microspheres)47 cultivation,
respectively.
Cells were incubated with 0.5mg/ml MTT or less / 15-60 min for measuring viability and metabolic
activity. For bulk assay of 25cm2 flasks the medium was decanted off carefully, 3ml of 16% Triton
X-100/0.01 NHCI added, shaken vigorously/1 min, standing for 30min48. Residual bubbles could be
most simply removed with a trace only of n-octanol before measuring with the microphotometer.
Cell counts were also obtained directly by detaching monolayers (trypsin/EDTA), neutralizing with
10-20% FBS in buffer, measuring cells per unit area (number of graticule eyepiece squares) or in a
16

hemacytometer or a 300l aliquot of cell suspension in a 24 well-plate, with or without trypan blue
viability test. In other cell viability counts with adherent cells 25cm2 flasks were lightly scratched
with 3x3 lines lengthways and widthways (9 cross-reference points) using the finest (size 0) sewing
needle in a holder held at an oblique angle to the outside surface. Each reference point could be
followed from 3 days to 1 month of an experiment with periodic changes of medium. Initial and
subsequent cell counts were made of 4-6-8 eyepiece graticule squares aligned around cross-points,
following the usual cell counting procedure of a hemacytometer. Following MTT incubation, alphaformazan crystal deposit were estimated and the mean cell density estimated relative to controls (no
drugs), the insolubility of the crystals being here an advantage. 5-10 reference point readings per
concentration of drug etc. A similar procedure was followed closely on 6-12-24 well-plates. Faster
and giving more information, cells were counted visually in a given area as above as total number
(TN), living (TLN) dead (TDN), blebbing, vacuoles etc (apoptotic) or necrotic, mitotic figures where
N-cell counts normalized to 100. This could be followed by lightly incubating with 25-50g
MTT/15-30 min incubation and scoring crystal density in each cell relative to a 0-10 sketched scale,
0 (no alph-formazan crystals) 10 (cell outline obscured by massive crystal deposit). Despite the
subjectivity of the latter, the precision was estimated at 5% sufficient for our purpose with 5+
counts per flask and when the MTT concentration/incubation time was adjusted to give the most
sensitivity on the 0-10 formazan crystal density scale.
Bacteria: Culture and procedures. The bacteria were incubated in 5ml liquid medium in closed
25cm2 cell culture flasks at 36-37C. The bacterial suspensions were discarded daily except for 0.10.5ml retained in a 1.0ml disposable syringe with a 20GA/2in needle (to prevent clumping) and used
in the inoculum in 5ml fresh medium in the same flasks and the various drugs were added. This
exposed the bacteria to a partly logarithmetic growth over 4-6 days. This was followed by a
reinoculating with fresh medium and withholding the drug and adding 120g/ml (net) of counterdrug
(5FC). The total bacteria was assayed by measuring the relative turbidity by the apparatus described,
17

using 0.1-0.25ml cell suspension in 5ml fresh medium. By diluting the bacterial suspension before
assaying the turbidity the growth could be measured beyond two orders of magnitude if necessary.
The persistence of the resistance (important to indicate presensitizing in the hospital) was also
measured by reinoculating as above without additives over a period of time, then assaying the
counterdrug.

18

Fig 1

Fig 2

Fig 3

19

Fig 4

20

Fig 5

21

Fig 6

22

Fig 7

23

Fig 8

24

Fig 9

Fig 10

25

Fig 11

26

Fig 12

ERRATUM, Fig 12
Omit Curve 3, since this refers to another test. The curve to legend 3, Fig 12 is fairly similar
to curve 2.
27