[CANCER RESEARCH 43, 3754-3758,
August 1983]
Comparable Growth Regulation of Five Human Tumor Cell Lines by
Neonatal Human Lung Fibroblasts in Semisolid Culture Media1
David Kirk,2 Susumu Kagawa,3 and Gudrun Vener
Huntington Medical Research Institutes, Pasadena, California 91101
ABSTRACT
Cellular growth interactions were studied between neonatal
human lung fibroblasts (NLF-13) and human tumor lines derived
from carcinomas of the prostate (PC-3, DU145), bladder (J82),
and endometrium (HEC-1A) and from a glioma (Hs 683t). NLF13 were interacted with tumor cells in soft agar or agarose media
using two experimental protocols. In one system, NLF-13 cells
were grown as anchored monolayers proliferating under the
tumor cell layer. In the second, NLF-13 were embedded directly
(nonanchored) into the agar or agarose layer with the tumor
cells. The results from both interaction systems were similar for
all five tumor lines. Anchored NLF-13 caused a dose-dependent
inhibition of tumor growth, whereas nonanchored cells produced
a dose-dependent growth stimulation. A time exposure experi
ment indicated that tumor stimulation and inhibition were biphasic
responses to NLF-13. It was concluded that low concentrations
of a diffusible NLF-13 product(s) accelerated tumor growth,
whereas high concentrations were inhibitory. Further, the pro
duction of the active NLF-13 substance(s) was positively corre
lated with NLF-13 growth rate. Tumor cell inhibition was irre
versible after a 5-day exposure to proliferating NLF-13 cells.
Another line of normal neonatal human lung fibroblasts (NLF147) showed inhibitory properties similar to those described for
NLF-13. However, preliminary studies with fibroblasts from the
skin of a Down's syndrome neonate (DS-172) and from a human
kidney tumor (KTF-130) have shown both these fibroblast types
to have a reduced ability to inhibit tumor cell cultures (J82)
compared to the neonatal lung fibroblasts (NLF-13 and NLF147).
INTRODUCTION
It has been found previously that neonatal human lung fibro
blasts (NLF-13) dramatically modified the growth of prostatic
carcinoma cells (PC-3) in culture (4, 5). Both of these studies
indicated a positive correlation between proliferation rate and
tumor cell inhibition. Whereas anchored (and proliferating) NLF13 inhibited PC-3 cells seeded in an agar overlayer, nonanchored
(and nonproliferating) NLF-13 suspended directly into the agar
markedly stimulated PC-3 growth (5).
The object of this study was to determine if these diverse
NLF-13 and/or PC-3 effects were spurious for this specific cell
combination or if they were more generally observed for other
fibroblast-tumor cell combinations in culture, (a) The effects of
NLF-13 were screened on a representative selection of carci
noma cell cultures derived from different tissues (prostate,
DU145; bladder, J82; endometrium, HEC-1A) to examine for
1Supported in part by the Jameson Foundation.
2 To whom requests for reprints should be addressed, at Huntington Medical
Research Institutes, 99 North El Molino Avenue, Pasadena, Calif. 91101.
3 Supported in part by Mr. Francis L. Moseley.
Received June 7, 1982; accepted April 21, 1983.
3754
tissue specificity of tumor cells. NLF-13 effects on a noncarci
noma tumor cell type was also investigated by studying a mesenchyme-derived glioma cell line (Hs 683t). (b) The specificity of
fibroblast effects was examined by comparing the effects of
normal neonatal lung fibroblasts (NLF-13) with abnormal fibro
blasts derived from the skin of a Down's syndrome neonate and
from a human kidney transitional cell carcinoma.
MATERIALS AND METHODS
Cells. Neonatal lung fibroblasts (NLF-13, NLF-147) were derived from
normal human lung tissue using standard expiant culture (7) and used at
passage levels 6 to 10. The established human tumor lines used were
derived from 2 prostatic carcinomas metastatic to both bone, PC-3 (3)
and brain, DU145 (12); an endometrial carcinoma, HEC-1A (6); a glioma,
Hs 683t (11); and a transitional cell carcinoma of the bladder, J82 (10).
The endometrial line, HEC-1A, was kindly provided by Dr. Jörgen Fogh
(Sloan-Kettering Institute of Cancer Research, Rye, N. Y.). Fibroblast
cultures were derived from the skin of a Down's syndrome neonate (DS172) and from a human kidney transitional cell carcinoma (KTF-130)
derived from a 72-year-old man using standard expiant culture (7). Both
DS-172 and KTF-130 were used at passage levels 4 and 1, respectively.
Agar and Agarose Cultures. The conventional agar bilayer method
used has been described previously (5). Soft (0.3%) and hard (0.5%)
Bacto-Agar (Difco Laboratories, Detroit, Mich.) or agarose (Seaplaque;
Marine Colloids, Rockland, Maine) were prepared with PFMR-44 (7) as
the nutrient base and supplemented with penicillin (100 ID/ml), Kanamycin
(100 M9/ml), and 20% FBS5 (Irvine Scientific, Santa Ana, Calif.). Cultures
were established in 60-mm Falcon Retri dishes by adding an overlay of
3 ml soft agar or agarose containing the tumor cells to a previously
prepared hard agar or agarose base (1.5 ml). The cultures were incubated
in a humidified 3% CO2 atmosphere at 36.5°. Cocultivation studies used
agar with PC-3 and HEC-1A inoculated at 5 x 103 per dish and agarose
with J82, Hs 683t, and DU145 inoculated at 2 x 10*, 2 x 10', and 10"
per dish, respectively.
As before (5), 2 culture systems were adapted to investigate the
effects of anchored and nonanchored NLF-13 cells on several human
tumor cell lines in either agar or agarose. In the anchored system, NLF13 cells were seeded in PFMR-4 containing 5% FBS in 60-mm dishes
and allowed to attach overnight. They were then covered with a hardlayer base which was in turn overlaid with the soft layer containing the
tumor cells. This arrangement precluded any NLF-13 and/or tumor cell
contact. In the nonanchored system, NLF-13 and tumor cells were mixed
and suspended in soft agar or agarose which was then added to a
prepared hard-layer base.
Coverslip Cultures. Coverslip cultures were prepared by inoculating
4.2 x 10s NLF-13 cells into 60-mm dishes containing a 40-mm glass
coverslip
in 4 ml of PFMR-4 medium supplemented
with 5% FBS.
Coverslips were used for cocultivation experiments 24 hr after seeding
by placing the NLF-13 cell surface of the coverslip directly on the agar
or agarose surface.
Scoring for CFE. Tumor growth was measured as CFE which was
4 As prepared in our laboratory.
5The abbreviations used are: FBS. fetal bovine serum; CFE, colony-forming
efficiency; Kf"-"1,inoculum of anchored NLF-13 cells producing 50% inhibition of
tumor cell colony-forming efficiency.
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Normal and Neoplastic Cell Interactions in Vitro
defined as the percentage of seeded cells that formed colonies. Colonies
were automatically enumerated and sized using a Hammamatsu image
analyzer. Only colonies greater than 90 um in diameter were counted
(average cell diameters of the human tumors used ranged from 15 to 20
urn). All cultures were set up in triplicate and scored for CFE between
10 to 20 days in culture.
Kinetic Analysis. Tumor growth dose-response curves were de
scribed in terms of 2 theoretical kinetic parameters, R,™»
and Km, devel
oped initially for enzyme kinetics but recently applied to clonal growth
data (8). The Ft™«
(maximal enzyme velocity) represented the maximal
CFE; the Km (half-maximal substrate concentration) represented the
number of NLF-13 cells producing half-maximal effects on tumor cell
growth. Both parameters were derived from statistically weighted regres
sions fitted to Lineweaver-Burk
plots. Tumor growth inhibition was
described in terms of the parameter KfLF which represented the number
of NLF-13 cells which produced 50% tumor cell inhibition. Since 100%
inhibition was reached for all tumor lines, the K["LFwas read directly from
the inhibition dose-response
curve.
RESULTS
Anchored versus Nonanchored NLF-13 Effects. The dra
matic difference between anchored and nonanchored NLF-13
effects on tumor cell growth is illustrated for the bladder carci
noma line J82 (Chart 1). The response of J82 to NLF-13 was
representative of all tumor lines studied. Whereas increasing the
NLF-13 cell inoculum resulted in a dose-responsive tumor cell
growth stimulation for nonanchored cells (Chart 1/\), anchored
NLF-13 caused marked tumor inhibition (Chart 18). These con
trasting NLF-13 effects on J82 cells closely resembled those
previously reported for PC-3 (5). The kinetics of these opposing
NLF-13 effects were compared for all 5 established human tumor
cell lines.
Comparative Kinetics of Nonanchored NLF-13 Tumor Stim
ulation. Growth-stimulatory effects of nonanchored NLF-13 were
compared for 5 human tumor lines (Table 1). The NLF-13 cells
induced maximal CFEs representing -fold increases of between
2.20 (Hs 683t) and 14.56 (J82). The sensitivity of the human
tumor cells to NLF-13 stimulation was conveniently expressed
as the ratio of KÕÕLF
(NLF cell inoculum eliciting half-maximal
response) to tumor cell inoculum (Table 1). Both the prostatic
carcinoma (PC-3) and the endometrial carcinoma (HEC-1 A) cells
were less sensitive than the other lines to growth stimulation by
nonanchored NLF-13. [Compare sensitivity values of PC-3 (17.0)
and HEC-1 A (25.0) with those of Hs 683t (2.5), J82 (1.56), and
DU145(4.0).]
Comparative Kinetics of Anchored NLF-13 Tumor Inhibition.
The effect of anchored NLF-13 on tumor CFE was compared for
the human tumor lines (Table 2). In all cases, inhibition was
observed and was expressed in terms of the parameter KfLF.
The sensitivity to NLF-13-inhibitory effects was standardized by
comparing the ratio of KINLFto tumor cell inoculum for each cell
line. Again, as for the growth-stimulatory effects, the PC-3 and
HEC-1 A lines were least sensitive to NLF-13 growth-inhibitory
effects. [Compare sensitivity values of PC-3 (8.0) and HEC-1 A
(7.0) with those of Hs 683t (0.25), J82 (0.3), and DU145 (0.2).]
Influence of Tumor Cells on NLF-13 Growth in Agarose. An
experiment was designed to determine if the increased CFE
observed in the mixed cultures of nonanchored NLF-13 and
tumor cells was due to stimulation of either the tumor cells or
NLF-13 cells, or both. Separate layers (1.5 ml) of tumor cells and
nonanchored NLF-13 cells in soft agarose were physically sep
arated by a hard agarose spacer layer (1.5 ml) and were prepared
using the original cell inocula which provided the maximal stim
ulation of CFE. None of the lines examined (DU145, J82, Hs
683t, HEC-1 A, and PC-3) induced NLF-13 colony formation in
agarose under these conditions. However, the CFEs of the tumor
lines were stimulated as observed before for the mixed cultures
and demonstrated that the growth stimulation in agarose was
unidirectional; NLF-13 stimulated the tumor cells and not vice
versa.
Table 2
Comparative kinetics of inhibition of human tumor cells by anchored NLF-T 3
cell in
ocula ratio produc
cell in
Tumor
celllinePC-36HEC-1
ing 100% inhibi
tion
mediumAgarAgarAgaroseAgaroseAgaroseK,""*40.00035.0005,0006,0002,000K,NLF:tumor
oculum8.07.00.250.30.2NLF:tumor
tion100551.250.780.5
AHs
683tJ82DU145Interac
X NLF CELLS
Chart 1. Effects of different inocula of anchored (•)
and nonanchored (O) NLF13 cells on the CFE of J82 in agarose. The J82 cells were seeded in agarose at 2
x 10*/dish (see "Materials and Methods") and scored for CFE after 13 days. Each
point represents the mean of triplicate cultures; bars, S.E.
NLF-13 cell inoculum producing 50% inhibition, determined directly from dosedependent inhibition curves.
6 Data taken from Kirk et al. (5).
Table 1
Comparative kinetics of stimulation of human tumor cells by nonanchored NLF-13
Tumor
linePC-3"
cell
HEC-1A
Hs 683t
J82
DU145Interaction
me
diumAgar
Agar
Agarose
Agarose
AgaroseCFE
cell
control6.87
(%)
±0.18C
2.50
0.50
0.90
4.40
(%)24.10
±0.60
8.72 ±0.24
±0.28
±0.03
1.10 ±0.20
±0.20
13.10 ±0.40
±0.60CFEÎSr 29.60 ±1.20-Fold
increase3.51
3.49
2.20
14.56
6.73KT85,000
inoculum17.0
125,000
50,000
31,250
40,000(C^rtumor
25.0
2.5
1.56
4.0
0 Data taken from Kirk ef al. (5).
c Mean ±S.E.
AUGUST 1983
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D. Kirk et al.
Irreversibility of NLF-13-induced Tumor Cell Inhibition. To
clarify whether the inhibition of tumor cells by anchored NLF-13
(DS-172), and from a transitional cell carcinoma of the kidney
(KTF-130). Marked differences existed between the growth-
was reversible, an experiment was set up which examined the
effect of varying the time of exposure to NLF-13 on subsequent
tumor cell viability. This was performed by placing inverted NLF13 coverslip cultures on soft agarose inoculated with 2.5 x 10"
PC-3 cells. Blank glass coverslips were placed on control PC-3
cultures. The NLF-13 coverslips were periodically removed and
all the PC-3 agarose cultures were photographed on Day 17
(Fig. 1). Compared to the control, PC-3 cultures exposed to NLF-
regulatory capacities of these fibroblast types. Although small
inocula of NLF-147 caused marginal growth stimulation, higher
cell inocula were found to cause a complete, dose-dependent
growth inhibition of J82 cells similar to the inhibition observed
previously for the other neonatal lung fibroblast line, NLF-13
(Chart 1). Nonanchored NLF-147, like nonanchored NLF-13 cells,
caused a marked growth stimulation of the tumor cells (data not
shown). On the other hand, fibroblasts from the Down's syn
13 for 2 to 4 days showed enhanced colony formation. However,
exposure to NLF-13 for 5 days and longer showed interesting
distributional changes. After a 5-day NLF-13 treatment, the PC3 cells centrally located beneath the NLF-13 were subsequently
unable to form colonies, whereas there was a marked nonrandom stimulation of CFE around the periphery of the NLF-13
coverslip resulting in a noticeable "ring" effect. After a 10- or 17-
drome patient showed an impressive biphasic growth regulation
of the tumor cells. Whereas cell inocula up to 105 caused a 3fold growth stimulation, subsequent increases in the DS-172 cell
inocula produced a gradual dose-dependent inhibition, 3 x 105
causing a 77% reduction in control levels. The KTF-130 cells
showed a still different effect. Although the NLF-147 (and NLF13) were generally associated with a dose-dependent inhibition,
the KTF-130 cells were observed to be exclusively stimulatory
day exposure to NLF-13, PC-3 was markedly inhibited, with the
limited growth observed being restricted to the periphery of the
NLF-13 coverslip.
Microscopic observation of inhibited tumor cells showed
marked cellular enlargement and degeneration. This NLF-13induced cytotoxic effect was specific for the tumor cells. An
chored NLF-13 monolayers did not produce a similar cytotoxic
effect on homologous NLF-13 coverslip cultures inverted on top
of the agarose surface.
Comparison of Different Anchored Fibroblasts on the
Growth of Bladder Tumor Cells (J82) in Agarose. Human
fibroblasts of different origin were compared for their effects as
anchored monolayers on the growth of bladder carcinoma cells
(J82) in agarose. Such comparisons were made (Chart 2) for
early-passage cultures of fibroblasts derived from neonatal lung
tissue (NLF-147), from the skin of a Down's syndrome neonate
to J82 carcinoma cell growth
studied.
in agarose for all cell inocula
DISCUSSION
The in vitro growth regulation of NLF-13 on a selection of
human carcinoma cells (DU145, J82, HEC-1 A) and human glioma
cells (Hs 683t) were essentially the same as that described
previously for the prostate carcinoma cells, PC-3 (5). All cell lines
demonstrated a dose-dependent growth stimulation in response
to nonanchored NLF-13, whereas similar inocula of anchored
NLF-13 caused a dose-dependent growth inhibition. However, 2
cell lines (PC-3, HEC-1 A) were less sensitive than the other lines
to both stimulatory and inhibitory NLF-13 effects. The ratios of
normal cell inocula to tumor cell inocula producing 50% inhibition
of tumor-cell CFE were much higher for both PC-3 (8:1) and
HEC-1 A (7:1) as compared with Hs 683t (0.25:1), J82 (0.3:1),
and DU145 (0.2:1). Similarly, the ratios of normal cell inocula to
tumor cell inocula producing 50% maximal stimulation of tumor
cell CFE were also much higher for PC-3 (17:1) and HEC-1 A
(25:1) than for Hs 683t (2.5:1), J82 (1.56:1), and DU145 (4.0:1).
These lowered sensitivities were not thought to reflect intrinsic
properties unique to PC-3 and HEC-1 A cells; rather, it appears
that the reduced response of these 2 lines was correlated with
the use of agar as the interaction medium, instead of agarose,
as was used with the other 3 cell lines. This is supported by the
observation (data not presented) that the ratio of normal cell
inocula to tumor cell inocula producing 50% inhibition for an
chored NLF-13 cells on PC-3 growth is reduced from 8:1 when
/•s
IO x FiBKOBLASTINOCULUM
Chart 2. Comparison of different anchored fibroblasts on CFE of bladder carci
noma cells (J82) in agarose. Anchored fibroblast cultures were set up as described
in "Materials and Methods" at a range of cell inocula. Bladder tumor cells (J82)
were added in the top agarose layer (5 x 10ddish) and scored for CFE after 13
days. Each point represents the mean of triplicate cultures; bars, S.E. Absolute
CFEs (%) for control cultures were 3.0 ±0.8 (DS-172), 7.3 ±0.8 (NLF-147), and
10.1 ±0.9(KTF-130).
3756
agar is used to 1.5:1 when agarose is used. Agarose does not
contain the sulfated polysaccharides which are known to be
growth-inhibitory components of agar (9). It is conceivable, there
fore, that it is this polyanion constituent of agar which to some
extent masked or modified the effect of the NLF-13 product(s)
on the tumor cell. Such modulations of the fibroblast effect by
agar components, and perhaps also by other exogenous defined
additives, may shed some light on the chemical nature of the
NLF-13 factor(s). A preliminary study (data not presented) using
dialysis membranes has already shown that the diffusible fibro
blast factor responsible for stimulation of tumor cells is greater
than M, 25,000. The macromolecular size of the fibroblast factor
should facilitate its subsequent isolation and purification.
The experimental data presented here support the previous
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VOL. 43
Normal and Neoplastia Cell Interactions in Vitro
contention (4, 5) that both NLF-13 effects were mediated by
diffusible substances. Two pieces of evidence further suggest
that both stimulation and inhibition of the tumor cells were
biphasic responses to an NLF-13 factor(s), the production of
which is dependent on NLF-13 growth rate, (a) A short exposure
(2 to 4 days) of PC-3 to anchored (or proliferating) NLF-13
stimulated growth, whereas longer periods (>5 days) caused
inhibition. After an intermediate exposure time (5 days), NLF-13
was observed not only to inhibit proximal PC-3 cells but also to
stimulate distal PC-3 cells within the same culture dish (Fig. 1).
The simplest explanation of this biphasic behavior of PC-3 is to
postulate the production of a single NLF-13 factor which acts in
a concentration-dependent
fashion; low concentrations stimu
late, whereas high concentrations inhibit in vitro tumor cell
growth, (b) NLF-13 monolayers that were reproductively inacti
vated with mitomycin C treatment stimulated PC-3 growth in
agarose instead of inhibiting them (data not presented) as pre
viously observed. Hence, proliferati ve status and not anchorage
per se was the causative NLF-13 factor determining the PC-3
cell response.
It is important to note that inhibition of PC-3 by NLF-13 was
observed to be irreversible after a 5-day exposure to anchored
NLF-13. Tumor cells thus treated were unable to form colonies
and were presumably nonviable. This irreversible inhibition made
it unlikely that the active fibroblastic substance was Interferon,
since interferon inhibitory effects were reported to be reversible
(1). In addition, interferon did not generally achieve 100% inhibi
tion in vitro (13) as was observed in this study.
This report has demonstrated that one normal neonatal human
lung fibroblast line (NLF-13) elicited similar growth-regulatory
effects on 5 different human tumor lines. This was not a spurious
effect associated with the NLF-13 cells per se since a second,
independently isolated line of normal neonatal lung fibroblasts
(NLF-147) also demonstrated similar growth-regulatory effects
on bladder carcinoma cultures. Both neonatal lung fibroblast
lines (NLF-13, NLF-147) produced inhibition of tumor cells as
anchored monolayers and stimulated tumor cell growth as
nonanchored cells. However, fibroblasts from 2 nonnormal tis
sues behaved very differently. Proliferating anchored monolayers
of Down's syndrome cells were able to both stimulate and inhibit
tumor cell growth and thus seem to occupy an intermediary
position between anchored monolayers of neonatal lung fibro
blasts (which are strong inhibitors of tumor cell growth) and
kidney tumor-derived fibroblasts (which are exclusive growth
stimulators). It is possible that these intermediary properties of
Down's syndrome fibroblasts are an in vitro reflection of the
predisposition that these individuals are known to have toward
cancers.
However, just what significance these results have in reference
to cancer in vivo is impossible to know. These simple interactional
studies involved only 2 cell types in a nonphysiological culture
matrix of agar or agarose. The in vivo tumor situation is inherently
more complex, involving other host cell types such as vascular
and hemopoietic cells. Of the latter type, macrophages have
been shown to have tumor-stimulatory effects in vitro (2). Al
though this culture system reported here is oversimplistic and
highly artificial, it affords a first step to begin dissecting out the
complex trophic cellular interactions that must exist between
tumor and host cells in vivo. In this respect, it will obviously be
important to further probe the initial differences observed be
tween fibroblasts derived from normal neonatal lung tissue and
those derived from pathological tissue.
ACKNOWLEDGMENTS
We thank Dr. M. E. Kaighn for kindly allowing us to use his cells and Laura
Hughes and Robert Olson for photographic assistance.
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D. Kirk et al.
CONTROL
NLF
TREATED (DAYS)
10mm
Fig. 1. Effect of varying the length of exposure to NLF-13 cells on the CFE of PC-3 in agarose. PC-3 cells were seeded at 2.5 x 10* cells/dish in agarose. Coverslip
cultures of NLF-13 (see "Materials and Methods") were then placed cells facing downward on the PC-3 agarose cultures. Blank glass coverslips were added as controls.
At Days 2, 4, 5, and 10, NLF-13 coverslips were removed; on Day 17, all PC-3 agarose cultures were photographed unfixed and unstained. Note: Day 17 shows a PC3 culture with an NLF-13 coverslip still intact, x 0.88.
3758
CANCER RESEARCH
VOL. 43
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Comparable Growth Regulation of Five Human Tumor Cell Lines
by Neonatal Human Lung Fibroblasts in Semisolid Culture
Media
David Kirk, Susumu Kagawa and Gudrun Vener
Cancer Res 1983;43:3754-3758.
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