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Tumor Biology |
Department of Internal Medicine IV, University of Heidelberg, 69115 Heidelberg, Germany [S. M.], and Department of Molecular Pharmacology and Toxicology, School of Pharmacy [E. C., S. M.], Department of Molecular Microbiology and Immunology, School of Medicine, and K. Norris Jr. Comprehensive Cancer Center [A. H. S.], University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To further determine the exact contribution of p21 to cell-regulatory events, cell lines without the p21 gene were established and investigated. In such studies, it was found that p21-negative mouse fibroblasts exhibit a partial deficiency in G1 cell cycle arrest after radiation or nucleotide depletion (11 , 12) . In human HCT116 colon carcinoma cells engineered to lack both copies of the p21 gene, a complete loss of G1 cell cycle arrest became evident, and this was associated with a predisposition to S- and M-phase uncoupling after exposure to DNA-damaging drugs (13) . Furthermore, an altered sensitivity of these p21-negative HCT116 cells to certain DNA-damaging chemotherapeutic drugs has been demonstrated both in vitro and in vivo (7 , 13, 14, 15, 16) . In addition, p21-negative cells have been shown to be defective in nucleotide excision repair, and it has been suggested that this may underlie their increased sensitivity to certain chemotherapeutic drugs (16) . However, despite the demonstrated role of p21 as a cell cycle inhibitor, inactivating mutations of this gene are very rarely found in tumor cells (17 , 18) , and mice lacking the p21 gene do not exhibit an increased rate of spontaneous tumors (11) . Therefore, the potential role of p21 in tumor development has remained largely unclear.
The in vitro culture of cells in suspension, where cells are allowed to form three-dimensional MCSs, is believed to more closely mimic in vivo conditions than the culture of cells in two-dimensional monolayers (19) . However, except for some hematopoietic cells, normal cells do not proliferate in suspension culture, a phenomenon called anchorage dependence. In contrast, tumor cells are able to continue to grow in suspension culture (or embedded in soft agar), and this anchorage-independent phenotype has been found to closely correlate with their ability to form tumors in animals (20 , 21) . In general, the growth rate in suspension culture varies greatly between different tumor cell lines (22) , which may indicate the function of cell type-specific processes modulating anchorage-independent growth. In this regard, it was shown in one study (23) that intercellular adhesions mediated by the cell surface receptor E-cadherin were required for the survival and anchorage-independent growth of human HSC-3 oral squamous carcinoma cells. In contrast, another study (24) described that transfected E-cadherin resulted in the suppression of anchorage-independent growth of EMT/6 mouse mammary carcinoma cells. Although the underlying basis for this discrepancy is unclear, the detailed analysis of E-cadherin in these processes is of importance because this molecule not only plays a crucial role in normal tissue morphogenesis and organization (25 , 26) but presumably also in invasive processes and the metastatic spread of tumor cells (27, 28, 29, 30) .
The contribution of CKIs to anchorage-dependent growth control has been recognized as well. After transfer of monolayer cells to suspension culture, the expression of several CKIs, such as p21WAF1, p27KIP1, p16INK4a, or p18INK4c, has been found elevated and implicated in the subsequent growth arrest of anchorage-dependent cells (22 , 31, 32, 33, 34, 35, 36) . Curiously, a very similar induction of p21 and p27 has also been detected in many anchorage-independent tumor cells (22 , 33) . It appears that in this latter case, the CKIs are sequestered in the cytoplasm and therefore prevented from exerting their inhibitory effect in the nucleus (33) . However, this neutralizing mechanism seems only partially effective because most, if not all, tumor cells in suspension exhibit reduced proliferation rates when compared with monolayer culture (22) . In addition, there are striking cell type-specific differences; in a review of 39 different tumor cell lines, only 22 were found to exhibit increased expression of either p16, p21, or p27 after transfer to suspension culture conditions (22) .
In an attempt to further explore the role of p21 in anchorage-independent growth, we comparatively analyzed the HCT116 human colon carcinoma cell line and derivatives thereof that were engineered previously (37) to lack both alleles of the p21 gene (HCTp21-/-). Here, we show that HCTp21-/- cells have lost the ability to grow independently of anchorage and die through apoptosis when transferred to suspension culture. This phenotype is attributable to the loss of induction of E-cadherin and insufficient cell-cell adhesion after detachment and can be overcome by transfection with either p21 or E-cadherin expression vectors. Moreover, HCTp21-/- cells exhibit severely reduced capacity for tumor formation in chicken embryos and are unable to form foci in monolayer culture. In addition, the absence of tight cell-cell interactions potentiates the sensitivity of these cells toward chemotherapeutic agents when assessed in suspension culture. Thus, our study links a major regulator of the cell cycle to the expression of a cell-cell adhesion molecule with implications for the tumorigenic potential and chemosensitivity of these cells. It further indicates that p21 is required for anchorage-independent growth, and tumorigenic potential, of HCT116 colon carcinoma cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Soft Agar Assays.
Assays were performed as described previously (38)
with
104 cells/well in a six-well plate.
Immunoblotting.
Immunoblotting was performed as described previously (35)
.
Monoclonal and polyclonal antibodies against E-cadherin were obtained
from Santa Cruz Biotechnology, Zymed, and ICN/Cappel. Antibodies
against p21 and polyclonal antibodies against P-cadherin and N-cadherin
were purchased from Santa Cruz Biotechnology. Anti-integrin antibodies
were a gift from P. C. Brooks (University of Southern California, Los
Angeles, CA).
Stable Transfection.
Human E-cadherin cDNA (39)
and p21 cDNA (40)
were stably transfected into HCTp21-/- cells using the vector
pcDNA6/V5-His (Invitrogen), which placed the gene under the control of
the CMV promoter. Stably transfected cells were selected with the
antibiotic blasticidin (Invitrogen).
Cell Cycle Analysis.
Cells from monolayer or suspension culture were harvested by
trypsinization and treated with 10 mg/ml hyaluronidase for 15 min at
37°C to disrupt cell-cell adhesions. Cells were then fixed in 90%
ethanol, treated with 40 µg/ml RNase A, stained with 20 µg/ml
propidium iodide, and analyzed by flow cytometry on a FACScan analyzer
(Becton Dickinson).
Oligonucleotide Treatment.
Synthetic phosphorothioate-modified oligonucleotides were provided by
the Core Facility of the K. Norris Jr. Comprehensive Cancer Center. The
sequences of anti-p21 oligonucleotides AS-MID and AS-IC, as well the
control oligonucleotides 18 MER and 21 MER, have been published
elsewhere and shown to be effective at inhibiting p21 expression
(41)
. The cytofectin GS2888 (Ref. 42
; a gift
from W. M. Flanagan, Gilead Sciences, Foster City, CA) was used for
transfection. One µM oligonucleotide was incubated with
105 cells/ml 24 h before detachment and
again immediately after detachment.
Cytotoxicity and Proliferation Assays.
Cell number and cytotoxicity were determined using an assay kit that is
based on the cellular conversion of a tetrazolium salt (MTT) into a
blue formazan product that is detected using a 96-well plate reader at
570 nm. The kit was obtained from Boehringer Mannheim and used
according to the manufacturer’s instructions.
TUNEL Assay.
To measure apoptotic cell death, cells were labeled with FITC, and
104 cells/point were analyzed with the use of
FACStar (Becton Dickinson). In addition, cell death was confirmed by
preparing cryosections of MCSs and staining of cells in
situ. For this purpose, MCSs of cells cultured in suspension for
48 h were collected, embedded in Tissue-Tek (Sakura), and frozen
on dry ice. Cryosections were air dried and fixed in 3% formalin.
TUNEL reaction (43)
was performed using a kit according to
manufacturer’s instructions (Boehringer Mannheim). After staining,
samples were processed for examination with a Nikon fluorescence
microscope using filters for FITC.
Annexin V Assay.
Externalized phosphatidylserine was detected by analyzing the binding
of FITC-labeled annexin V (44)
with the use of a
commercially available kit (Calbiochem). Cells were costained with
propidium iodide and analyzed by flow cytometry for necrosis and
apoptosis.
E-Cadherin Blocking Antibody.
Cells were seeded at a density of 105 cells/ml in
96-well plates in the presence of mouse anti-human E-cadherin
monoclonal antibody (clone SHE78-7; Zymed Laboratories) at 1 µg/ml
and incubated for up to 72 h. A mouse anti-p53 monoclonal antibody
added to monolayer and suspension cultures at up to 10 µg/ml was used
as control.
Chicken Embryo Tumorigenicity Assay.
The assay was performed as reported previously (45)
with
the following modifications. Tumor cells at a density of
106 cells/40 µl were transferred onto the
chorioallantoic membrane of 10-day-old chicken embryos. The eggs were
further incubated for 7 days, and the tumors were harvested. Tumor
volume (V) was calculated using the formula
V = d2
D/2, where d is the smallest tumor diameter and
D is the largest diameter (in millimeters).
Adhesion Assays.
Adhesion assays were performed according to procedures described
recently (46)
with some modification as follows. HCTp21 or
HCTp21-/- cells (4 x 104) were
seeded onto 96-well microplates coated with different ECM proteins. The
wells had been coated with up to 50 µl of ECM protein solution until
evaporation had finished with the following concentrations: 25 µg/ml
vitronectin in PBS, 25 µg/ml fibronectin in PBS, 25 µg/ml laminin
in PBS, 0.1 mg/ml collagen in 0.2 N acetic acid, and 200
µg/ml BSA in serum-free medium. After coating, all wells were rinsed
three times with serum-free medium containing 200 µg/ml BSA. The
cells were allowed to adhere for 15–30 min (depending on the ECM
protein) in a 5% CO2 incubator. Incubation time
was stopped as soon as cells started to adhere. The wells were then
washed three times with serum-free medium to remove unattached cells.
Cell number was determined using the MTT assay as described above.
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RESULTS |
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. FACS analysis indicated that the slower growth of
HCTp21-/- cells was not attributable to cell cycle blockage, because
the percentage of these cells in S phase was 34% as compared with 20%
for the HCT116 cells (Fig. 3A)
. This is in agreement with earlier observations that
HCTp21-/- cells have a reduced ability to arrest in
G1 of the cell cycle (13)
.
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. Greatly increased apoptotic cell death of
HCTp21-/- cells as compared with HCT116 cells became obvious at
48 h after detachment and increased further over the course of
several days. At day 7, more than half of the whole population of
HCTp21-/- cells were apoptotic, whereas only 12% of HCT116 cells
stained positively (Fig. 3B)
. Apoptosis as the main cause of
cell death in HCTp21-/- cells was further confirmed with the use of
the FITC-labeled annexin V, which binds to phosphatidylserine of
apoptotic cells (not shown), as well as by in situ TUNEL
staining of cryosections from MCS of each cell type (not shown).
Cell-Cell Adhesion Is Crucial for the Survival of HCT Cells in
Suspension Culture.
To investigate whether the pronounced differences in MCS formation were
involved in the induction of apoptosis, we used the enzyme
hyaluronidase to break and prevent cell-cell interactions
(47)
. When added to suspension cultures, hyaluronidase
completely prevented cell-cell aggregation of both HCT116 and
HCTp21-/- cells (Fig. 2B)
and caused cell death in either
cell type (Fig. 2A)
. This was observed at enzyme
concentrations that were nontoxic per se, i.e.,
that neither interfered with the attachment of these cells to cell
culture plates nor their growth rate in monolayer culture (not shown;
Ref. 47
). Therefore, these results indicated that the
ability of HCT116 cells to form compact MCSs allowed them to evade cell
death.
MCS Formation of HCT Cells Is Mediated by Induction of E-Cadherin.
Cell-cell interactions by cadherins have been shown recently to
modulate survival of tumor cells under anchorage-independent conditions
(23)
. Therefore, studies were undertaken to assess
expression and function of cadherins. Using immunoblotting techniques,
a striking variance in the expression of E-cadherin in suspension
culture cells was found. In HCT116 cells, the amount of E-cadherin
protein was strongly increased when cells were transferred from
monolayer to suspension culture over a period of 48 h (Fig. 4A)
. In comparison, HCTp21-/- cells exhibited a
substantially weaker increase in E-cadherin. Two other cadherins,
P-cadherin and N-cadherin, were not detected in either cell type (not
shown), which is consistent with their preferred expression in
different groups of cells. The expression of members of the integrin
family (
v-ß3 and v-ß5), which are cell surface proteins that
determine cell-matrix interactions, was not significantly altered in
either cell type (Fig. 4A)
. Additionally, the binding of
cells before and after suspension culture was quantitated with a
functional assay using plates coated with individual ECM proteins. No
difference in adhesion to these distinct matrices was observed between
the two cell types (Fig. 4B)
, which indicates the absence of
alterations in these types of cell-matrix interacting proteins. Thus,
these results further emphasize the specificity of differential
E-cadherin induction.
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Importantly, HCTp21+/- cells, which retained one allele of the
p21 gene, had similar basal levels of E-cadherin as well and
exhibited an induction somewhat weaker than HCT116 cells (Fig. 4C)
. In suspension, these cells formed MCSs with slightly
less density than the HCT116 cells (not shown).
To determine whether E-cadherin was indeed the crucial determinant of
the observed cell-cell interactions, HCT116 cells in suspension were
incubated with a monoclonal antihuman E-cadherin antibody (clone
SHE78-7). This antibody had been shown before to perturb the function
of E-cadherin and prevent cell-cell aggregation of tumor cells
(23)
. Indeed, when added to HCT116 cells, this antibody
completely blocked the formation of compact MCSs. The microscopic
appearance of these cells was essentially identical to the appearance
of nonantibody-treated HCTp21-/- cells as shown in Fig. 1
,
i.e., there were weakly aggregated spheroids of small size
and with individual cells clearly visible (not shown). The presence of
this antibody did not affect cell growth or morphology of these cells
in monolayer culture. As a control, an unrelated monoclonal antibody
was used. In this case, even at up to 10-fold higher concentrations, no
effects on spheroid formation were observed. Moreover, the stable
transfection of E-cadherin cDNA into HCTp21-/- cells conferred the
ability to form dense MCSs with the same morphological appearance as
those formed by HCT116 cells (Fig. 6
, Lane 4). Taken
together, these results indicate that the increased expression of
E-cadherin is the major determinant of MCS formation in these cells.
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, cells treated with p21-antisense oligonucleotides
exhibited decreased E cadherin expression after cellular detachment,
whereas control oligonucleotides had no inhibitory effect. Upon
microscopic inspection, it became apparent that the cells treated with
p21-antisense oligonucleotides were unable to form compact MCSs (not
shown), and cell death was observed leading to significant differences
in cell number (Fig. 5B)
. Thus, the HCT116 cells treated
with p21-antisense oligonucleotides displayed a phenotype that was very
similar to HCTp21-/- cells.
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, Lane 1. The other five clones did not harbor elevated
levels of p21 and did not exhibit increased levels of E-cadherin nor
formation of dense MCSs. Thus, 80% of p21-transfected clones (four of
five) displayed elevated levels of E-cadherin and at the same time had
gained the ability to form dense MCSs. Mock-transfected cells exhibited
the same phenotype as the respective parental cells (i.e.,
no alterations), thus excluding transfection per se as the
cause of the observed alterations. Therefore, the above experiments
demonstrate a causative link between the expression of p21 and the
induction of E-cadherin in suspension cells.
Loss of p21 Potentiates Chemosensitivity in Suspension Culture.
Differences in the sensitivity toward anticancer drugs between HCT116
and HCTp21-/- cells grown as monolayer have been reported previously
(13)
. Moreover, in other tumor cells, it has been
indicated that cell-cell adhesion may affect cellular chemoresistance
as well (48)
. Therefore, to assess the impact of loss of
p21 on the chemosensitivity of detached cells, we incubated suspension
cultures of HCT116 and HCTp21-/- cells with either daunorubicin, a
clinically used anticancer drug, or with
3,6-diaziridinyl-1,4-benzoquinone (DZQ), a quinoid model
compound commonly used to study the redox-cycling abilities of
anticancer quinones (49)
. The addition of either drug to
cells immediately at the onset of suspension culture (before spheroid
formation) revealed a 2- and 2.1-fold higher chemosensitivity of
HCTp21-/- cells, which was comparable with what we observed in
monolayer culture. However, when the drugs were added 24 h after
the onset of suspension culture (when spheroids had formed), the
difference was 10- and 26.7-fold, respectively (Fig. 7)
. Therefore, in suspension culture, the differences in chemosensitivity
between these two cell types were greatly enhanced, indicating that p21
might contribute to this process via its effects on the expression of
E-cadherin and spheroid formation.
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, HCT116 cells were strongly positive in all of these three assays. In
contrast, HCTp21-/- were negative; they did not form foci, they
lacked the ability to grow independently of anchorage, and they formed
a >10 times smaller tumor mass in chicken embryos (Fig. 8)
. Therefore,
our results clearly indicate a correlation between the loss of the
p21 gene and a greatly reduced tumorigenic potential in
these cells.
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DISCUSSION |
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We found, however, that upon transfer of these cells to suspension
culture conditions, obvious differences became apparent.
Microscopically, there was a striking difference in their morphological
appearance. Whereas HCT116 cells form compact, dense MCSs, HCTp21-/-
cells aggregate only very loosely (Fig. 1)
. This difference is extended
to their proliferative capacity, because HCT116 cells continue to
proliferate independently of anchorage (for at least 3 weeks in
soft agar), whereas HCTp21-/- cells cease proliferation in suspension
and die through apoptosis within 5 days. Thus, our finding was
quite surprising, because it suggested "pro-tumorigenic" functions
of the cell cycle inhibitor p21. This interpretation seems rather
contradictive, because p21 has been characterized as a major
executioner of p53-induced cell growth arrest and tumor suppression
(1
, 40
, 50
, 51)
. Interestingly, however, in contrast to
p53, which is very frequently found mutated, p21 mutations are
extremely rare in human tumors (17
, 18)
. Furthermore,
whereas p53-knockout mice develop spontaneous tumors (52
, 53)
, this phenomenon is not observed in p21-knockout mice
(11)
. Additionally, in experimental mouse skin
carcinogenesis assays, it was shown that the genetic deletion of p21
enhances the formation of papillomas but not their malignant conversion
(54)
. From these studies, it appears that the contribution
of p21 to tumorigenesis is clearly distinct from p53 and may be
restricted to early phases of neoplastic development.
Intriguingly, comparable to our results with p21, a "paradoxical
tumor inhibitory effect of p53 loss" has been described
(55)
. In this study, p53-knockout mice were crossed with
transgenic mice expressing the oncogenes v-Ha-ras,
v-fos, or transforming growth factor targeted
to the epidermis. In keeping with the established role of p53 as a
tumor suppressor, it was anticipated that the resulting progeny would
display further increased hyperplasia and papillomatogenesis in
response to various insults such as phorbol ester treatment or
wounding. However, contrary to expectation, it was found that these
mice, now harboring the respective oncogene in the background of a
p53-negative genome, exhibited a block of spontaneous, phorbol
ester-promoted, and wound-induced hyperplasia (55)
.
Although the processes underlying these surprising effects are unclear,
it was speculated that cell type specificities, differentiation states,
or a critical combination of the synergistic neoplastic insults may
play a role. In this regard, it is interesting to note that Li-Fraumeni
individuals, who are hemizygous with respect to p53 deletions, have a
high spectrum of sarcomas, lymphomas, and breast tumors but only few
epidermal tumors (56)
. Alternatively, it was suggested
that there may be a requirement for p53 expression during the early
stages of skin tumor development, e.g., during the response
to phorbol ester-promoted or wound-induced stimuli
(55)
.
In comparison with our results and others discussed above, it could be
hypothesized that p21 may be required during later stages of tumor
development, at least in certain types of tumors, as exemplified by the
HCT116 cell line. Such a requirement of p21 could be attributable to
its documented apoptosis-inhibitory function (41
, 57, 58, 59, 60, 61)
,
which is indeed observed in the present study; when HCT cells with and
without p21 are transferred to suspension culture, HCT116 cells
continue to proliferate independently of anchorage, whereas HCTp21-/-
cells die through apoptosis (Figs. 2
and 3)
.
Our studies further demonstrate that the antiapoptotic effects of p21
during suspension culture of HCT116 cells appear to be mediated via the
induced expression of the cell surface molecule E-cadherin and closely
correlate with the ability of the cells to form dense MCSs. This is
indicated by the following observations: (a) expression of
E-cadherin is induced in HCT116 but not in HCTp21-/- cells after
detachment (Fig. 4A)
; (b) in the presence of
anti-E-cadherin antibodies, HCT116 cells loose the ability to form
dense MCSs and die through apoptosis (not shown); (c)
overexpression of transfected E-cadherin in HCTp21-/- cells results
in dense MCS formation and anchorage-independent growth (Fig. 6)
.
Together, these results indicate that MCS formation and
anchorage-independent proliferation of HCT116 cells is dependent on the
expression of E-cadherin. In this regard, our results are in agreement
with findings by others (23)
, who established that in
HSC-3 human squamous carcinoma cells, E-cadherin-mediated
intercellular adhesions were required for anchorage-independent growth
and survival of cells in suspension.
In addition, we show that the induced expression of E-cadherin is
dependent on the presence of p21: (a) E-cadherin is induced
in HCT116 but not in HCTp21-/- cells after detachment (Fig. 4A)
; (b) in HCT116 cells, inhibition of p21 by
antisense-p21 oligonucleotides results in the concurrent reduction of
E-cadherin expression during suspension culture (Fig. 5A)
;
(c) transfection of p21 cDNA into HCTp21-/- cells restores
elevated expression of E-cadherin (Fig. 6)
. Taken together, the above
observations strongly indicate that p21 regulates anchorage-independent
growth and survival via the increased expression of E-cadherin. It
should be noted that although HCTp21-/- cells do exhibit reduced
capability for DNA repair (16)
, it is unlikely that the
lack of E-cadherin regulation in these cells is attributable to
unrecognized mutations in the E-cadherin gene:
(a) we have used two different HCTp21-/- cell lines that
were selected independently of each other (37)
;
(b) the level of E-cadherin expression is the same in
HCT116, HCTp21+/-, and both HCTp21-/- cell lines during monolayer
culture (Fig. 4C)
, indicating that the basal level
regulation is intact; and (c) transfection of HCTp21-/-
with p21 cDNA restores the elevated expression of E-cadherin in
suspension culture (Fig. 6)
.
A further finding of our analysis is the greatly enhanced
chemosensitivity of HCTp21-/- cells in suspension culture.
Differential chemosensitivity of HCT116 and HCTp21-/- cells to
anticancer agents have been described before (13)
.
However, those studies used only cells grown as a monolayer. In our
present investigation, we confirm that HCTp21-/- cells are
significantly more sensitive than HCT116 cells when grown as monolayer;
however, when transferred to suspension culture, this difference is
greatly augmented, for example, from 2.1- to 26.7-fold for daunorubicin
(Fig. 7)
. Because the ability of suspension cells to form MCSs via
E-cadherin has been proposed to condition their sensitivity to
chemotherapeutic drugs (48
, 62)
, it is conceivable that
the differential expression of E-cadherin in our HCT cell model is
responsible for the observed effect. Furthermore, because we show that
elevated E-cadherin expression is dependent on the presence of p21, our
results further indicate that in suspension culture, p21 might
determine cellular chemosensitivity, at least in part, via the
expression level of E-cadherin.
The indicated involvement of p21 in the chemosensitivity of suspension culture cells is related to results by others (63) , implicating another CKI, p27KIP1, in the resistance of tumor cells to anticancer agents. Similar to our findings with p21, it was established that the down-regulation of p27 expression with antisense oligonucleotides in suspension culture resulted in reduced intercellular adhesion and increased cellular sensitivity to the drug 4-hydroperoxycyclophosphamide.
Overall, however, it appears that the exact contribution of E-cadherin and of p21 and p27 to anchorage-independent cellular growth is strongly dependent on the cell type under investigation. For example, E-cadherin has been characterized as a major growth suppressor of EMT/6 mouse mammary carcinoma cells in suspension culture (24) , whereas in other studies it was shown to be required for anchorage-dependent proliferation and survival of HSC-3 human squamous carcinoma cells (23) . Similarly, the induction of p21 or p27 that has been observed after transfer of cells from monolayer to suspension culture can be detected in many but not all cell types (22 , 33 , 35) . It is likely that differences in the genetic background contribute to the observed discrepancies between different cell types. Thus, for the future analysis of these processes, it would be helpful to comparatively investigate more cell lines where defined genes of interest have been deleted by targeted disruption.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Research Grant
CN-82601 from the American Cancer Society (to A. H. S.) and by Grant
1RO1HL53467 (to E. C.) and Grant R29CA74278 (to A. H. S.) from the
NIH. S. M. is a Feodor-Lynen-Fellow of the Alexander-von-Humboldt
Foundation. 
2 To whom requests for reprints should be
addressed, at Department of Molecular Microbiology and Immunology, 2011
Zonal Avenue, HMR-405, Los Angeles, CA 90033-1034. Fax:
(323) 442-1721; E-mail: schontha{at}hsc.usc.edu
3 The abbreviations used are: CKI,
cyclin-dependent kinase inhibitor; MCS, multicellular spheroid;
poly-HEMA, poly-(2-hydroxyethyl methacrylate); MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL,
terminal deoxynucleotidyl transferase-mediated nick end labeling; ECM,
extracellular matrix; FACS, fluorescence-activated cell sorter.
Received 5/24/99. Accepted 10/27/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cancer Res., 56: 4413-4423, 1996.This article has been cited by other articles:
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) were treated with either daunorubicin
or with DZQ for 24 h, and cell viability was determined
thereafter with the MTT assay. On the left, the drugs
were added at the time of transfer from monolayer to suspension culture
(0 h detachment); on the right, the drugs were added
24 h after the onset of suspension culture (24 h detachment). The
IC50 ratio of HCT116 over HCTp21-/- cells is given as
R, which indicates the difference in chemosensitivity
between the two cell lines. Each point reflects the average of eight
measurements; bars, SD.