THE ROLE OF APOPTOSIS IN THE INHIBITION OF A SECONDARY TUMOR BY
CONCOMITANT RESISTANCE IN A MOUSE MODEL OF METASTASES
MARCELA FRANCO, PEDRO BEKINSCHTEIN, PEDRO D. di GIANNI,
OSCAR D. BUSTUOABAD, SILVIA VANZULI, RAUL A. RUGGIERO
Instituto de Investigaciones Hematológidas, Academia Nacional de
Medicina, Buenos Aires
Key words: apoptosis, concomitant resistance, matastasis
of tumor-bearing mice to a second tumor challenge, that is,
concomitant resistance, was studied using the LB tumor model. In a
secondary LB tumor inhibited by concomitant resistance an increase in
the percentage of apoptotic cells and alterations in cell-cycle
distribution were observed. Similar alterations in cell-cycle
distribution were observed. Similar alterations were observed in LB
tumor cells incubated with serum from tumor- bearing mice. The data
presented in this paper suggest apoptosis as one of the mechanisms
involved in tumor dormancy due to concomitant resistance.
rol de la apoptosis en la inhibición del tumor secundario por la
concomitante en un modelo murino de metastasis. La resistencia
concomitante es la capacidad de un individuo portador de un tumor, de
inhibir el crecimiento de un segundo implante tumoral. En este trabajo
se estudia el rol de la apoptosis en este fenómeno utilizando como
modelo el tumor murino LB. Se observó, en el tumor secundario LB
inhibido por resistencia concomitante, un aumento en el porcentaje de
células apoptóticas y alteraciones en el ciclo celular. Se
obtuvieron resultados similares cuando las células del tumor LB
fueron indubadas con suero de ratones portadores del
mismo tumor. Los resultados sugieren que la apoptosis está
involucrada en el estado de tumor dormido característico del
fenómeno de resistencia concomitante.
Received: 19-VI-1996 Accepted:
Postal address: Dr. Marcela Franco, Instituto de
Investigaciones Hematológicas, Academia Nacional de Medicina, P. Melo
3081, 1425 Buenos Aires, Argentina
Concomitant resistance is the phenomenon according to which a
tumor- bearing host inhibits the growth of a secondary implant of the
same tumor at a distant site1. The relevance of this model is that it
can mimic the relationship between a primary tumor and its distant
metastases2, 3. Concomitant resistance induced by weakly and
non-immunogenic tumors has been described as non- specific and
mediated by a mechanism presumibly unrelated to any known conventional
immunological mechanisms4, 5, 6, 7. In previous papers8, 9, we have
demonstrated that BALB/c mice bearing a non-immunogenic lymphoma,
called LB, generated a strong concomitant resistance. We have also
demonstrated that serum from these LB-bearing mice had an inhibitory
activity (not attributable to cytotoxic antibodies) on in vitro (3H)
thymidine incorporation by LB-tumor cells.
This activity was proportional to the intensity of concomitant
resistance and was mediated by factor/s of low molecular weight
(1000-1200 D). There is a growing body of evidence on the importance
of apoptosis in tumor inhibition induced by chemotherapeutic drugs and
radiation10, 11 and by antiangiogenic factors12.
The aim of this paper was to investigate whether apoptosis and cell
cycle alterations were involved in the concomitant resistance induced
by LB tumor. The role of apoptosis was evaluated a) in vivo in the
secondary LB tumor implant and b) in vitro in LB cells incubated with
serum from LB tumor-bearing BALB/c mice (LB serum).
BALB/c mice were s.c. inoculated with 106 LB tumor cells and were
with 105 LB cells in the opposite flank seven days later. The control
group received this "second" implant only.
The percentage of cells with hypodiploid DNA content (apoptotic) in
the secondary 7-day tumor was investigated using hypotonic Propidium
Iodide (PI) fluorochrome staining and cytofluorometric technics as
previously described byothers13, 14 and was confirmed by DNA
electrophoresis and by fluorescence microscopy.
The secondary LB tumor, prevented to grow by concomitant resistance,
showed a significantly higher percentage of apoptotic cells (18.1 ±
2.1) than that observed in the control group (3.9 ± 1.3) (mean ±
SE). A representative histogram of DNA content is shown in Figure 1.
Similarly, a significant increase in the apoptotic population in LB
tumor cells incubated with different dilutions of LB serum during 7
hours was observed (Table 1) as compared with incubation in normal
serum. Additional evidence of apoptotic cell death was obtained by gel
electrophoresic analysis of the DNA degradation products, showing the
typical "ladder" pattern and by fluorescence microscopy
(data not shown).
A correlation between cell cycle alterations and apoptosis has been
reported in various systems; in some, apoptosis occurs only after cell
cycle arrest and in others, cells are preferentially susceptible to
apoptosis at specific phases of the cell cycle10, 11. In this paper,
we analyzed the cell cycle distribution of the non- apoptotic
population of the secondary LB tumor and determined the existence
ofdifferences between experimental and control groups. In effect,
there was a decrease in the percentage of cells in the G2-M phases and
an increase in the proportion of cells in the S phase in the second
tumor implant in mice bearing a primary LB tumor as compared with the
control group (experimental group: G0- G1: 66.7 ± 1.4, S: 29.7 ±
0.6% (P < 0.001) and G2-M: 3.5 ± 1.3% (P < 0.001); control
group: G0-G1: 75.6 ± 3.9%, S: 10.9 ± 4.9% and G2-M: 9.8 ± 1.1%)
(Mean ± SE 0f 3 assays).
The non-apoptotic population of LB tumor cells incubated with LB serum
also showed alterations in cell-cycle distribution: a significant
decrease in the percentage of cells in G2-M compartment was observed
(Table 1), in accordance with data obtained in in vivo experiments.
The lower porportion of cells in G2-M obtained both in in vivo and in
in vitro essays are in accordance with previous data showing a
decrease in the number of metaphases per field at the site of the
secondary tumor implant7.
Although the data presented here do not allow us to explain the
alterations observed in cell cycle distribution, some considerations
can be made. A special susceptibility to apoptosis of cells in G2-M
(and likely, in G0-G1) could account for these results. On the other
hand, we cannot discard that LB cells could go to apoptosis
independently of the position in the cell cycle. In this case, the
changes observed in the cell cycle could be the consequence of growth
arrest that makes it difficult for LB cells to complete the S stage
and pass through G2, raising the possibility that a
"cytostatic" effect of LB serum could exist independently of
its apoptotic effect.
Up to now, the identity of the factor/s responsible for the observed
apoptosisneeds to be established. The results reported herein suggest
that apoptosis is involved in the quiescence or dormancy observed in a
secondary LB tumor inhibited by concomitant resistance and also
suggest that serum factors from LB- tumor bearing mice- previously
reported as associated with the phenomenon of concomitant resistance8,
9 --could play a role in the observed apoptosis.
Acknowledgements: We are grateful for helpful discussions
with Isabel Piazzón, Irene Nepomnaschy and Christiane Dosne
Pasqualini. We thank Antonio Morales and Juan José Portaluppi for
excellent technical assistance. This work was supported by grants from
ILEX-CONICET and from FUNDALEU
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Fig. 1. Flow cytometric DNA fluorescence profiles of PI-stained LB
tumor cells from a secondary LB tumor and its control in a
representative experiment. Arrow indicates the population of nuclei
with hypodiploid DNA content (apoptotic).
TABLE 1.-- Apoptosis and alterations in cell cycle distribution in
LB- tumor cells incubated in vitro with different dilutions of LB
serum or normal serum.
serum dilution % apoptotic % viable
cells cells in each phase
G0-G1 S G2-M
1:2 30.6 ± 4.7* 62.5 ± 5.1 32.1 ±
5.8 5.3 ± 1.8*
LB serum 1:4 18.9 ± 3.5* 60.9 ± 6.0 34.2
± 2.0 4.8 ± 1.1*
1:8 14.7 ± 5.8 ND ND
normal 1:2 14.0 ± 2.5 62.9 ± 3.9 25.5 ±
3.2 11.7 ± 1.2
serum 1:4 11.0 ± 2.5 66.1 ± 3.6 22.7 ±
2.7 12.2 ± 1.4
1:8 10.6 ± 3.2 ND ND
LB tumor cells were incubated in vitro with different dilutions of
LB serum or normal serum in a 7-hour assay. Incidence of apoptotic LB
tumor cells treated with RPMI was 6.83 ± 1.4%. Percentages of LB
tumor cells incubated with RPMI: G0G1: 71.5 ± 4.7; S: 18.5 ± 6.2;
G2-M: 10.0 ± 2.8. (*: P < 0.001;