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`
`
`PHIGENIX
`PHIGENIX
`Exhibit 103 1
`Exhibit 1031
`
`

`

`Differential Killing Efficacy of Twenty Antitumor Drugs on
`Proliferating and Nonproliferating Human Tumor Cells
` 
`Benjamin Drewinko, Myra Patchen, Li-Ying Yang, et al.
`Cancer Res  
`
`1981;41:2328-2333.
`
`Updated version
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`[CANCER RESEARCH 41, 2328-2333,
`0008-5472/81
`/0041-OOOOS02.00
`
`June 1981]
`
`Differential Killing Efficacy of Twenty Antitumor Drugs on Proliferating
`and Nonproliferating Human Tumor Cells1
`
`Benjamin Drewinko,2 Myra Patchen, Li-Ying Yang, and Barthel Barlogie
`
`of Laboratory Medicine [B. D., L. Y. Y.] and Developmental
`Departments
`Anderson Hospital and Tumor Institute, Houston, Texas 77030
`
`Therapeutics
`
`[M. P., B. B.], The University of Texas System Cancer Center, M. D.
`
`ABSTRACT
`
`The lethal effects of a 1-hr treatment with 20 antitumor drugs
`on proliferating
`and nonproliferating
`cultured
`human colon
`carcinoma cells (line LoVo) were analyzed quantitatively by the
`colony-forming
`technique.
`Proliferating
`cells were obtained
`from exponentially
`growing
`cultures, while nonproliferating
`cells were from cultures in a stationary phase of growth. The
`1-hr treatment was intended to approximate serum peak levels
`after bolus administration.
`Two agents, c/s-platinum and vin-
`desine, were more effective on nonproliferating
`than on prolif
`erating cells. Mitomycin C, nitrosoureas,
`and dihydroxybisal-
`kylanthracenedione
`were equally effective on proliferating and
`nonproliferating
`cells. The low lethal activity «1 log) of meth-
`ylglyoxal
`bis(guanylhydrazone),
`hycanthone,
`and vinblastine
`was similar
`in proliferating
`and nonproliferating
`cells. For most
`drugs (Adriamycin,
`rubidazone,
`bleomycin, maytansine,
`vin-
`cristine, epipodophyllotoxin,
`fluorouracil,
`hydroxyurea, meth-
`otrexate, and transplantinum)
`cytotoxicity was significantly less
`pronounced (or even totally absent)
`in nonproliferating
`than in
`proliferating
`cells. These results demonstrate the significance
`of cellular proliferation
`kinetics
`in determining
`sensitivity
`to
`antitumor
`therapy. Nonproliferating
`human cells have de
`creased sensitivity
`to most antitumor agents. An occasional
`agent may present
`increased activity to nonproliferating
`cells;
`but at best,
`few agents can be expected to be as effective on
`nonproliferating
`as on proliferating
`cells.
`
`INTRODUCTION
`
`Cell cultures provide a rapid, efficient, and economical assay
`system for cytotoxicity
`screenings of antitumor agents, allowing
`elucidation
`of
`the mode of action of a drug in a controlled
`systematic fashion with a high degree of resolution. However,
`a major drawback of tissue culture experiments
`resides in the
`difficulty of extrapolating
`information obtained on exponentially
`growing cells to the expected responses of tumor cells in vivo
`where large fractions of the population may be in the quiescent
`state (Go cells)
`(43). Quiescent
`cells are usually considered
`less sensitive than proliferating
`cells to the lethal activity of
`most antitumor drugs (3, 6, 9, 17, 37, 52, 54-56).
`Recent
`investigations
`suggest
`the usefulness of utilizing sta
`tionary-phase
`cultures as an adequate in vitro model of in vivo
`neoplasias with low fractions of proliferating
`cells (low-growth
`fraction)
`(2, 3, 24, 32). Stationary-phase
`cultures are obtained
`by allowing cells to replicate without
`refeeding until such time
`when no net
`increment
`in cell numbers is demonstrated.
`This
`can result either
`from cessation of multiplication
`(with elonga
`
`times) or from a balance between cell
`tion of cell cycle transit
`death and cell multiplication (i.e.,
`the number of cells born per
`unit
`time equals the number of cells disintegrating
`per unit
`time). Some investigators
`have studied the lethal activity of
`antitumor agents on this type of culture system using cells of
`rodent origin. They reported
`considerable
`differences with
`respect
`to the response obtained for exponentially
`growing
`cultures (4, 5, 44, 46) and suggested that
`these differences
`might be exploited at the clinical
`level (49).
`We evaluated the lethal efficacy of a variety of antitumor
`agents on an established colon carcinoma cell
`line of human
`origin while in the exponential and in the stationary phases of
`growth. Our
`results demonstrated
`that,
`in contrast
`to studies
`effected on rodent cells, human cells in the stationary phase of
`growth have decreased sensitivity to most antitumor agents.
`
`MATERIALS AND METHODS
`
`Cell Line. The cells used in this investigation were from a
`carcinoembryonic
`antigen-producing
`colon
`carcinoma
`line
`(LoVo cells) established in 1972 (12). Cells are maintained as
`monolayer cultures in Ham's F-10 medium supplemented with
`
`20% fetal calf serum, vitamins, glutamine, and antibiotics. LoVo
`cells generate glandular
`structures
`in vitro when grown as
`monolayers and in vivo when propagated as xenografts in nude
`mice (14). The generation time of exponentially
`growing cells
`is 29.3 hr, and the transit
`times through each stage of the cell
`cycle are: Gì,14.7 hr; S, 10.7 hr; and rG2 + M, 4.8 hr. Single
`cells plated in fresh medium give rise to large colonies with a
`PE3 ranging from 35 to 70% (14).
`Growth Kinetics Characteristics. To establish the time se
`quence of
`the in vitro growth of LoVo cells (i.e., exponential
`and stationary phase), aliquots of 10s, 5 x 105, and 7 x 105
`cells were seeded into sets of 60-mm Petri dishes containing
`5 ml of growth medium and maintained without
`refeeding
`throughout
`the span of the experimental
`interval. Every 24 hr
`for 21 days, 2 replicate cultures from each set were harvested
`using techniques
`described
`previously
`(12) and were proc
`essed for cell counting, LI determinations,
`FCM studies, and
`colony formation. Cell counts were performed with the aid of
`an electronic
`particle counter
`(Model ZBI Coulter Counter;
`Coulter Electronics,
`Inc., Hialeah, Fla.). For LI determinations,
`the cells were pulse-labeled
`for 30 min with [3H]dThd (1 juCi/
`ml; specific activity, 3.0 Ci/mmol) before harvesting. Cytocen-
`trifuge preparations were processed for radioautography
`using
`a 50% solution of llford emulsion (Polysciences,
`Inc., Warring-
`ton, Pa.) in distilled water, exposed for 2 weeks, and developed
`
`' Supported by Grants CA 23272, CA 14528, and CA 16763 from National
`Cancer
`Institute, Department of Health, Education, and Welfare.
`2 To whom requests for reprints should be addressed.
`Received July 21,1980;
`accepted March 5, 1981.
`
`used are: PE, plating efficiency; LI, labeling index; FCM,
`3 The abbreviations
`thymidine; BCNU, 1,3-bis(2-chloroethylM-n¡trosourea;
`flow cytometry;
`dThd,
`BLEO, bleomycin;
`c/s-DDP, c/s-diamminedichloroplatinum;
`VDS, deacetyl
`blastine amide sulfate.
`
`vin
`
`2328
`
`CANCER RESEARCH VOL. 41
`
`
`
`Downloaded from on April 19, 2014. © 1981 American Association for Cancer Research. cancerres.aacrjournals.org
`
`
`
`

`

`Cell Killing of Proliferating and Nonproliferating
`
`Cells
`
`Table 1
`List of antitumor agents used to treat proliferating
`
`and nonproliferating
`
`
`
`CommonnameAdriamycin
`
`cells
`No.123127
`
`249992301739
`AMSADHAQ.CINSC
`
`name1
`4-Hydroxydaunorubicin
`Methanesulfon-m-anisidide,
`AMSAAnthracenedioneChemical
`4'-<acridinylamino)
`{ {2-{(2-
`1,4-Dihydro-5.8-bis
`hydroxyethyl)amino]ethyl)
`amino) 9.1 0-anthracene-
`dione dihydrochlorideAbbreviationADR
`BLEO
`BCNU
`
`Bleomycin
`Carmustine
`
`Cisplatin
`
`Epipodophyllotoxin
`
`5-Fluorouracil
`Hycanthone
`Hydroxyurea
`Maytansine
`Methotrexate
`
`Methyl-GAG
`
`Mitomycin C
`Rubidazone
`
`Transplatin
`
`Vinblastine
`Vincristine
`Videsine
`
`-ni-
`
`1,3-Bis(2-chloroethyl)-1
`trosourea
`4-[3-<2-Chloroethyl)-3-nitro-
`soureido]-cis-cyclohex-
`anecarboxylic
`acid
`c/s-Diamminedichloroplas-
`tinum
`4'-Demethylepipodophyllo-
`toxin 9-<4,6-O-ethylidene-
`ß-D-glucopyranoside)
`
`Glutamic acid, N-{{p-{[(2.4-
`diamino-6-
`ptendinyl)methyl]methy-
`lamino)benzoyl}}
`Methylglyoxal
`bis(guanylhydrazone)
`
`benzoylhydra-
`Daunorubicin
`zone hydrochloride
`frans-Diamminedichloroplas-
`tinum
`
`Deacetyl Vinblastine amide
`sulfate
`
`125066
`409962
`
`119875
`
`141540
`
`19893
`142982
`32065
`153858
`740
`
`c/s-Acid
`
`c/s-DDP
`
`VP-16-213
`
`5-FU
`
`HU
`MAYT
`MTX
`
`Methyl-GAG
`
`32946
`
`Mito C
`RUB
`
`frans-DDP
`
`VBL
`VCR
`VOS
`
`26980
`164011
`
`49842
`67574
`245467
`
`in Kodak D19 (Eastman Kodak, Rochester, N. Y.). FCM studies
`were
`conducted
`on mithramycin-ethidium
`bromide-stained
`cells using a Phywe ICP II pulse cytophotometer
`(Phywe AG,
`Gottingen, Germany)
`(14). For PE determinations,
`aliquots of
`200 cells/dish were dispensed into 60-mm Petri dishes,
`incu
`bated for 21 days, stained, and fixed with 2% crystal violet
`in
`95% ethanol, and the colonies were counted with a stereomi-
`croscope. PE was defined as the ratio of colonies to the number
`of plated cells.
`Survival Assay. Stock cultures were harvested and counted
`with the aid of a Model ZBI Coulter Counter electronic particle
`counter. Cell suspension
`aliquots were seeded into 60-mm
`Petri dishes (5 x 105 cells/dish).
`The cells were incubated at
`37°in a 5% CO2 atmosphere in air for 48 to 72 hr to achieve
`exponential growth and for 8 days without medium replenish
`ment
`to achieve a stationary
`growth phase (see "Results").
`
`and the cells were exposed to
`The medium was discarded,
`for 1 hr at 37°. The drug was
`increasing drug concentrations
`decanted, and the cells were washed twice in Hanks' balanced
`
`im
`suspension
`salt solution, harvested as a monodispersed
`mediately after
`the treatment using hyaluronidase
`(102 Ill/ml)
`for 5 min followed
`by trypsin (2.5%)
`for 5 min, and then
`counted. Known aliquots of the cell suspension were dispensed
`into 60-mm Petri dishes so that 50 to 100 colonies would
`appear after 21 days of
`incubation
`in a 5% CO2 humidified
`atmosphere at 37°.The colonies were stained with 2% crystal
`violet
`in 95% ethanol. Viability was defined as the ability of
`single cells to give rise to a colony of >50 cells.
`In each
`experiment,
`the PE of at least 6 control cultures was assessed
`simultaneously. Control cultures,
`both in exponential
`and in
`stationary
`growth phases,
`consisted
`of cells treated in the
`same manner as the test cells but without
`receiving drug. The
`survival
`fractions
`for
`the different drug concentrations were
`normalized with respect
`to the individual
`controls
`for each
`experiment. All experiments were repeated at least
`twice with
`triplicate samples for each drug concentration.
`Because in many instances the shape of the survival curve
`obtained for cells in stationary phase differed substantially from
`that determined for exponentially
`growing cells,
`it was impos
`sible to quantify differences
`in the degree of cell kill by con
`ventional parameters (13). Therefore, we arbitrarily calculated
`differences
`in efficacy as the ratio of the survival
`levels deter
`mined at the midrange point of concentrations
`used for each
`drug.
`Drugs. All drugs (listed in Table 1) were obtained from the
`Cancer Chemotherapy Evaluation Branch, Division of Cancer
`Treatment, National Cancer
`Institute. Drug solutions were al
`ways prepared in growth medium immediately before an ex
`periment, and the pH was adjusted if necessary to 7.2 to 7.4.
`Water-soluble drugs were first dissolved in 0.9% NaCI solution.
`Lipid soluble drugs were first dissolved in pure ethanol or in
`20% ethanol-80% propylene glycol.
`In some cases (epipodo-
`phyllotoxin,
`methanesulfon-m-anisidine,
`4'-(acridinylamino),
`etc.),
`the drug was dissolved first with the furnished solvent. At
`the final concentrations
`used in our experiments, each solvent
`used alone failed to affect
`the viability of either exponential or
`stationary-phase
`cells.
`
`RESULTS
`
`Proliferation
`
`characteristics
`
`of LoVo cells inoculated
`
`at 3
`
`refeeding were
`different cell densities and maintained without
`analyzed. After a lag time of 24 to 36 hr, doubling times were
`independent of initial
`inocula and ranged from 34 to 38 hr with
`a mean value of 36.3 hr. After about 2 doublings (3 to 4 days),
`the initially higher cell
`inocula (5 and 7 x 105) entered a phase
`of stationary growth as defined by no increments
`in cell num
`ber. The plateau segment of the growth curve lasted for about
`4 days after which time cell death increased, as reflected by a
`decrease in cell numbers with a halving time of 103 hr. The LI
`declined from 33% during exponential growth to 1% just at the
`time cultures entered stationary phase. However, simultane
`ously FCM-determined DNA-dependent
`compartment distribu
`tions revealed that about 15% of the cell population had an S-
`phase DNA content and about 16% of the cells had a G2-phase
`DMA content. Stationary phase could not be reverted to expo
`nential growth by: (a) replacement
`of supernatant with fresh
`medium;
`(b) brief
`incubation with trypsin and reincubation with
`the same supernatant;
`or (c) harvesting and transferring
`the
`entire cell population to new dishes containing fresh medium.
`Only when cells were replated in fresh medium at a density
`lower
`than 6.45 x 10" cells/sq
`cm (<1.5
`x 106 cells/dish)
`in
`was logarithmic
`growth resumed. No significant
`variations
`PE as a function of stage growth (exponential,
`stationary, or
`decline) were noted. The fluctuations
`in PE were similar
`to
`those documented for experiments conducted at different
`times
`and with different batches of stock cells.
`for
`On the basis of
`these results, survival was compared
`exponentially growing LoVo cells (2 to 3 days after subculture)
`and cells in stationary
`phase (8 days after subculture),
`cis-
`Diamminedichloroplatinum
`was more effective (efficacy
`ratio,
`
`JUNE
`
`1981
`
`2329
`
`
`
`Downloaded from on April 19, 2014. © 1981 American Association for Cancer Research. cancerres.aacrjournals.org
`
`
`
`

`

`S. Drewinko et al.
`
`2.3) on ce\\s in stationary phase than on exponentially growing
`ones (Chart 1). This effect
`resulted from the abrogation of the
`shoulder
`region of
`the survival
`curve, while the slope was
`maintained essentially intact. Vindesine was also more effective
`on cells in stationary phase (efficacy ratio of 18.9), but
`in this
`case the shape of the survival curve was similar
`for both cell
`classes.
`and vin-
`hycanthone,
`bis(guanylhydrazone),
`Methylglyoxal
`blastine had a similar
`low killing effect on both exponentially
`growing and stationary-phase
`cells (Chart 2). 1,4-Dihydro-5,8-
`bis{{{2-[(2-hydroxyethyl)amino]ethyl}amino}}9,10-anthra-
`cenedione
`dihydrochloride
`(data not shown), mitomycin C,
`BCNU,and4-[3-(2-chloroethyl)-3-nitro-ureido]-c/s-cyclohex-
`anecarboxylic
`acid (Chart 3) displayed
`the same powerful
`effect on both cell classes.
`Agents
`considered
`to be cell cycle sensitive and to act
`primarily on cells positioned
`in the S phase of
`the cycle (5-
`fluorouracil, methotrexate,
`and hydroxyurea) were consider
`ably less lethal on stationary-phase
`cells (Chart 4; Table 2)
`than on exponentially growing cells.
`In fact, methotrexate com
`pletely failed to kill cells in the stationary
`phase of growth.
`
`100
`
`Cis-DDP
`
`VINDESINE
`
`10
`
`a
`(0
`
`•Exponential growth
`- °Stationary phase
`
`0.1
`
`10
`
`20
`
`15 0
`/»g/ml
`LoVo cells treated with
`and nonproliferating
`Chart 1. Survival of proliferating
`increasing concentrations
`of cis-DDP or VDS for 1 hr. In this, and in subsequent
`charts, points are mean values of at least 2 separate experiments,
`each with 3
`replicates per concentration.
`Bars, S.E.
`
`40
`
`60
`
`80
`
`100
`
`BCNU
`
`CIS-ACID
`
`MITO C
`
`\.
`
`•Exponential growth
`°Stationary phase
`
`0
`
`10
`
`20
`
`30
`
`40 0
`
`20
`
`40
`
`60
`
`80 0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`LoVo cells exposed to
`and nonproliferating
`Chart 3. Survival of proliferating
`alkylating agents for 1 hr. Bars, S.E. cis-Acid, 4-[3-(2-chloroethyl>-3-nitrosour-
`eido]-c/s-cyclohexanecarboxylic
`acid; mito C. mitomycin C.
`
`5-FU
`
`MTX
`
`HU
`
`K
`
`K\,
`
`•Exponential growth
`? Stationary
`phase
`
`0
`
`1000
`
`2000 0
`
`1000
`
`ng/ml
`
`2000 0
`
`200
`
`400
`
`600
`
`800 1000
`
`LoVo cells treated for 1
`Chart 4. Survival of proliferating and nonproliferating
`hrwith S-phase-sensitive
`agents. Bars. S.E. 5-FU. 5-fluorouracil; MTX, glutamic
`acid, W-{(p-{[2,4-diamino-6-pteridinyl)methyl]methylamino}benzoyl();
`HU, hy-
`droxyurea.
`
`Table 2
`Differential efficacy of antitumor drugs on proliferating
`cellsDrugs5-FU6
`LoVo
`
`and nonpro/iferating
`
`MTX
`HU
`VCR
`MAYT
`VP-16
`ADR
`RUB
`AMSA
`frans-DDP
`BLEOStationary/exponential81.3
`a Ratio of survivals at midrange dose point.
`For definition of abbreviations, see Table 1.
`
`1.91.3
`
`2.5
`2.6
`3.2
`2.5
`3.3
`3.8
`3.0
`8.0
`
`Mitotic inhibitors [vincristine, maytansine, and 4'-demethylepi-
`podophyllotoxin
`9-(4,6-O-ethylidene-/3-D-glucopyranoside)]
`all
`had a similar
`type B survival curve (13), and in all
`instances
`their efficacy on stationary-phase
`cells was about 2.5- to 3-fold
`less than that on exponentially
`growing cells (Chart 5; Table
`2). Similar differences
`in efficacy
`between exponential
`and
`stationary-phase
`cells were observed
`for DNA-intercalating
`agents such as anthracycline
`derivatives
`(adriamycin,
`dauno-
`rubicin benzoylhydrazonehydrochloride)
`and methanesulfon-
`m-anisidide, 4'-(acridinylamino)
`(Chart 6). The ineffective frans
`isomer of diamminedichloroplatinum
`was even less active when
`
`METHYL-GAG
`
`VINBLASTINE
`
`HYCANTHONE
`
`growth
`•Exponential
`°Stationary phase
`
`200
`
`SO
`
`Mg/ml
`
`50
`
`LoVo cells exposed to
`and nonproliferating
`Chart 2. Survival of proliferating
`increasing concentrations
`of methylglyoxal bis(guanythydrazone),
`(methyl-GAG).
`vinblastine, and hycanthone for 1 hr. Bars, S.E.
`
`2330
`
`CANCER RESEARCH VOL. 41
`
`
`
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`
`
`
`

`

`Cell Killing of Proliferating and Nonproliferating Cells
`
`100
`
`so
`
`10
`
`i
`
`1
`
`0.5
`
`0.1
`
`BLEO
`
`•Exponential
`; Stationary
`
`growth
`phase
`
`10
`
`20
`
`30
`
`40
`
`50
`
`eg/ml
`
`•Exponential growth
`°Stationary phase
`
`10
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50 0
`
`0.2
`
`0.4
`
`0.6
`
`0.8
`
`1.0 0
`
`LoVo cells treated for 1
`and nonproliferating
`Chart 5. Survival of proliferating
`hr with mitotic inhibitors. Bars, S.E. VP-16, 4'-demethylepipodophyllotoxin
`9-
`(4,6-O-ethylidene-/?-D-glucopyranoside).
`
`RUBIDAZONE
`
`AMSA
`
`•Exponential
`' Stationary
`
`growth
`phase
`
`0.2
`
`0.4
`
`0.6
`
`0.6 0
`
`0.2
`
`0.4
`
`0.6
`
`0.6
`
`1.0 0
`
`0.2
`
`0.4
`
`06
`
`08
`
`1 0
`
`LoVo cells treated for 1
`and nonproliferating
`Chart 6. Survival of proliferating
`hr with DMA-intercalating
`agents. Bars, S.E. AMSA, methanesulfon-m-anisidide,
`4'-(acridinylamino).
`
`evaluated with stationary cells (data not shown). The antibiotic
`BLEO also showed markedly
`less activity on the stationary-
`phase cells than on exponentially
`growing ones (Chart 7).
`
`DISCUSSION
`
`the increased
`concerning
`agreement
`There is generalized
`tumors to most anticancer
`sensitivity
`of
`rapidly proliferating
`regimens with respect
`to the inadequate response shown by
`tumors with low-growth fractions (10, 48, 54, 58). These clin
`ical conclusions are supported by a vast array of experimental
`evidence obtained in a variety of systems ranging from bacterial
`and mammalian cell cultures to transplanted rodent
`tumors (3,
`8, 9, 15-17,
`20, 28, 29, 36-38,
`41, 42, 52, 55-57).
`The
`difference
`in cell-killing
`efficacy is generally attributed to the
`ability of quiescent cells to repair drug-induced damage before
`it becomes a fixed,
`lethal event at mitosis (21, 22, 25, 34, 38,
`39) and can sometimes be overcome by massive doses of the
`antitumor agent (53).
`However,
`in certain cell types (most notably cultured tumors
`of rodent origin) and for a restricted range of agents,
`increased
`sensitivity
`on nonproliferating
`cells with respect
`to that of
`exponentially
`growing elements has been reported (4, 6, 23,
`33, 36, 44).
`In most such instances, observations obtained in
`one cell system were not supported
`by other
`investigators,
`even when using similar cell
`types. Thus,
`the reported
`in
`creased efficacy of BCNU (4, 6), BLEO (4) or hydroxyurea (33)
`on nonproliferating
`cells was not confirmed by others in differ-
`
`for proliferating
`Chart 7. Survival
`1 hr with BLEO. Bars, S.E.
`
`and nonproliferating
`
`LoVo cells treated for
`
`ent or even similar cultured cell systems (3, 17, 33, 45, 50,
`56). These discrepancies
`can be attributed in part
`to species-
`related biological differences. More important, perhaps,
`is the
`diversity
`in methodology
`used to obtain nonproliferating
`cul
`tures (2, 3, 17, 24, 32, 35, 47). This diversity
`can generate
`significant
`differences
`in the physiological
`properties
`of
`the
`treated cells and in their metabolic disposition
`of the noxious
`agent (16, 20, 24, 26, 32) and can also lead to differences
`in
`sensitivity and clonogenicity
`as a function of
`the duration of
`stationary growth (32, 51). Another equally important possibil
`ity resides in the fact
`that
`the stationary-phase
`cultures used
`by different
`investigators
`differ significantly
`in their cell cycle
`compartment
`distribution.
`Thus,
`it
`is possible that purported
`differences
`in drug sensitivity of stationary versus proliferating
`cells may actually reflect
`the sensitivity of cells accumulated in
`different
`stages of the cell cycle. For instance, Bhuyan ef al.
`(6) and Barranco ef al. (5), using uptake of [3H]dThd, showed
`a decreased proportion of S-phase cells in plateau cultures (79
`to 45% for L1210 cells and 62 to 4% for Chinese hamster
`ovary cells). Bhuyan further documented
`this reduction in S-
`phase cells by FCM, although he also showed an increased
`proportion of cells in G2 + M. Barranco ef al. showed that
`in
`their unfed plateau cultures most cells were blocked in a G,-
`like phase after 90 to 120 hr, although 1 to 4% of the population
`continued
`to incorporate
`[3H]dThd. Tobey and Ley (47) and
`Madoc-Jones
`and Bruce (31),
`respectively, demonstrated
`that
`Chinese hamster and L-cells were arrested in the Gìstage of
`the cycle during
`stationary
`phase, whereas Thatcher
`and
`Walker
`(45), Drewinko ef al. (11 ), Macieira-Coelho
`(30), and
`Ross and Sinclair
`(40) showed considerable
`accumulation
`in
`G2 phase for hamster embryo, human myeloma, human embryo
`fibroblasts, and Chinese hamster cells,
`respectively.
`the cell
`For our LoVo cells in the stationary phase of growth,
`cycle stage compartment distribution analyzed by FCM showed
`a considerable
`proportion of cells with S-phase (15%) and G2
`+ M (16%) DNA content, although the LI was only 1%. These
`results indicate that mammalian cells in the stationary phase of
`growth do not necessarily accumulate in d and that
`failure to
`
`JUNE 1981
`
`2331
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`
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`

`

`B. Drewinko et al.
`
`[3H]dThd cannot be considered evidence of non-
`incorporate
`S-phase position. Apparently, many cells in stationary phase
`can be delayed in the G2 phase of the cycle (18, 19, 30, 45),
`while others are stopped in the midst of synthesizing DMA or
`synthesize DNA very slowly.
`As shown by others for occasional agents, our human culture
`system also disclosed 2 drugs (c/s-DDP and VDS) that were
`more effective on stationary-phase
`cultures than on exponen
`tially growing cells.
`In a manner similar
`to that observed by
`Hageman ef al. for BCNU on mouse plasmacytoma cells (21),
`the increased effectiveness
`of c/s-DDP on the human tumor
`cells resulted from the abrogation of the shoulder
`region of the
`survival
`curve while the slope was retained
`intact. Such
`changes in the shape of the survival curve can also be observed
`in exponentially
`growing LoVo cells if c/s-DDP is administered
`at 41 °(1 ), suggesting that the capacity to absorb drug-induced
`damage without expressing
`a lethal effect
`is thermosensitive
`and modulated
`by the proliferative
`status of
`the LoVo cells.
`VDS was almost 20 times more effective on stationary-phase
`cells than on exponentially
`growing ones.
`It is difficult
`to rec
`oncile this observation with the lethal mode of action ascribed
`to Vinca alkaloids (i.e.,
`inhibition of mitotic spindle). Thus,
`it is
`possible either
`that VDS has an additional
`killing mechanism
`not shared by its congeners or that the activity of this particular
`agent persists long enough to inhibit division when the treated
`cells in stationary phase are allowed to proliferate for colony
`formation.
`In the manner described by others for alkylating agents (7,
`27, 45),
`the efficacy of nitrosourea derivatives and mitomycin
`C was quite similar
`for both exponentially growing and station
`ary-phase LoVo cells. This similar efficacy could originate from
`an intrinsic property of the interaction of alkylating agents and
`the target cells or result
`from the inability of stationary-phase
`cells to repair potentially lethal damage before they are manip
`ulated into exponential growth (25).
`Most clinically used antitumor drugs displayed the decreased
`efficacy on LoVo cells in the stationary phase of growth that
`is
`so often seen in other cell systems. The decreased efficacy
`was noted for cell cycle-active
`agents,
`for mitotic inhibitors,
`and for DNA-intercalating
`agents. Of particular
`interest, BLEO
`was about 8 times less active on the human tumor cells in
`stationary phase of growth than on the exponentially
`growing
`counterpart.
`Many agents displayed a type B or D survival curve pattern
`when applied to exponentially growing cells reflecting the pres
`ence of a fraction of
`less sensitive cells (13). Although these
`patterns
`could represent
`cell cycle stage-dependent
`differ
`ences in sensitivity,
`they may well be the result of a significant
`proportion of nonproliferant
`cells present
`in the exponentially
`growing population, as previously documented for LoVo cells
`(14).
`in sensitivity between expo
`The reasons for the differences
`nentially growing and stationary-phase
`cells may be multiple,
`including differential drug transport
`rates, ability to repair sub-
`lethal and potentially
`lethal damage,
`target availability,
`etc.
`Therefore,
`it is possible that some such differences in sensitivity
`may be attenuated or completely abolished if exposure to the
`agent
`is prolonged for periods longer
`than 1 hr or if the cells
`are allowed to repair some drug-induced
`damage before har
`vesting for exponential growth into colony-formation.
`In conclusion, our results reiterate the significance of quan
`
`potential
`kinetics for determining
`tifying cellular proliferation
`in contrast
`sensitivity to tumor
`therapy and demonstrate
`that,
`to cells of
`rodent origin, nonproliferating
`human cells have
`decreased sensitivity to most antitumor agents when treatment
`is short
`lived. Although a rare drug may show increased activity
`on nonproliferating
`cells, usually only agents that display al
`kylating properties can be expected to be only as effective on
`proliferating
`as on nonproliferating
`cells.
`
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