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Apoptosis and Response to Radiation: Implications for Radiation Therapy

Apoptosis and Response to Radiation: Implications for Radiation Therapy

ABSTRACT: Apoptosis is a mode of cell death that is currently of intense research interest in developmental and cancer biology. For more than 40 years, radiobiologists have been aware of cells in irradiated specimens that display the features of apoptosis. Despite this knowledge, however, it has proved difficult to elucidate the role of apoptosis in end points important in radiation therapy and to distinguish its role from that of other modes of cell death, namely, reproductive cell death. In this article, the evidence relevant to this question will be critically reviewed. Conclusions that have significant implications for radiation therapy will be drawn to suggest that tumor cells with apoptotic propensity are more sensitive to radiation. If this hypothesis is confirmed, strategies can be envisioned that may restore apoptotic propensity to radioresistant tumor cells for therapeutic benefit. [ONCOLOGY 11(3):349-356, 1997]

Introduction

A poptosis, or programmed cell death, is characterized by specific features
that allow it to be distinguished from necrosis in tissues.[1] This distinction
is necessary because, whereas apoptosis occurs as part of many normal biologic
processes, especially during the development of an organism, it may also
play a critical role in certain pathologic conditions, including cancer.[2]

The importance of apoptosis in cancer biology has been generally appreciated
only in recent years, and this has led to a surge of reports of its occurrence
in various normal and tumor cell systems treated with radiation, drugs,
or biologicals. Although these reports clearly demonstrate that apoptosis
occurs in model cell systems in response to cytotoxic treatments, the relative
impact of apoptotic cell death on tumor response to therapy is essentially
unknown. This is due largely to the fact that most studies to date have
focused on the assessment of apoptosis in cultured cells. Apoptosis in
human tumors following therapy has not been systematically evaluated due
to technical and logistical constraints.

Nonetheless, research on radiation-induced apoptosis has uncovered new
fundamental concepts relating to the basic processes responsible for cell
death following irradiation and, most important, is beginning to reveal
the details of the biochemical and molecular pathways that regulate these
processes. This knowledge will most likely lead to entirely new strategies
for modulating radiation-induced cell killing in tumors or normal tissues
for therapeutic advantage.

The ability to cure a tumor with radiation may depend on various factors,
including tumor cell proliferation kinetics, tumor hypoxia, the number
of tumor clonogens, and intrinsic tumor cell radiosensitivity.[3] Apoptosis
may play a role in more than one of these factors, but, because apoptosis
is known to occur in cells irradiated in vitro, the most obvious role is
that apoptotic propensity may dictate intrinsic tumor cell radiosensitivity.
Interest in understanding the mechanistic basis for intrinsic cell radiosensitivity
has been stimulated by reports that tumor types whose cells display radioresistance
in culture are associated with treatment failures.[4] However, the question
of what determines a cell's intrinsic radiosensitivity remains a central
issue in radiobiology.

As discussed in a recent review, the "classic" view held that
the end point relevant to radiation therapy was "reproductive cell
death," which results directly from unrepaired DNA strand breaks.[5]
Radiobiologists now recognize the need to reevaluate this classic view
and consider new concepts, such as effects on cellular membranes, signal
transduction pathways, induction of gene expression, cell-cycle regulation,
oxidative stress processes, and the influence of growth factors. The need
to reevaluate the role of various pathways of cell death in response to
radiation has also been recently appreciated. Thus, whereas for many years
radiobiologic research focused exclusively on reproductive cell death,
much current effort is also being directed toward understanding the role
of apoptosis.

This article will briefly review our current understanding of the role
of apoptosis in radiation response and will then discuss the implications
of this role in radiation therapy.

Model Systems

In Vitro Cell Systems

In vitro cell systems have been used extensively in radiobiology since
1955, when Puck and Marcus[6] developed clonogenic assays for cell survival
using cultured mammalian cells. This important advance provided a model
system for the study of the relationship of basic cellular processes that
modify radiation response (eg, DNA damage and repair mechanisms) to loss
of cell reproductive capacity. Although the knowledge gained using these
cell systems has been critical to our understanding of the effects of radiation
on cells, such cell systems necessarily focused investigators' attention
on reproductive cell death, to the detriment of research centered on other
modes of cell death, such as apoptosis.

The term "apoptosis" was coined by Kerr et al in 1972 to describe
a process of programmed cell death distinct from necrosis.[1] Whereas apoptosis
has become a "hot" area of research during the last few years,
radiobiologists have been aware of it for at more than 40 years. Prior
to 1972, what we now refer to as apoptosis was included in a process called
"interphase death," a term that referred to the death of cells
before their first postmitotic division. Fundamentals of Radiobiology,
a classic textbook written by Bacq and Alexander and published in 1961,[7]
clearly distinguishes interphase death from reproductive cell death, which
is termed "mitotic death."

The definitions of Bacq and Alexander[7] do not distinguish between
necrosis and apoptosis, however. That distinction is one of the major contributions
made by Kerr et al in 1972.[1] One can find numerous reports from the 1950s
and early '60s describing cellular responses to radiation identical to
apoptosis. One of these, a set of cinemicrographic observations by Schrek[8]
published in 1955, provides unequivocal photographic illustrations of rabbit
lymphocytes undergoing apoptosis within 4 hours after 10 Gy of irradiation.

Apoptosis vs Secondary Apoptosis--The rather large number of
papers published during the last few years reporting apoptosis in irradiated
cultured cell systems has possibly led to some misconceptions about the
role of the apoptotic process in radiation response. In addition to the
occurrence of apoptosis as part of interphase cell death, apoptosis has
been reported in cultured cell systems in which the primary mode of death
is reproductive cell death. In these latter cases, apoptosis occurs after
the first postirradiation mitotic division, and therefore, may have consequences
that differ from those occurring when cells die before the first postirradiation
division.

This issue has been discussed in detail in another review,[9] but some
additional clarification may be warranted. Throughout the remainder of
this article, I will refer to cell death as "apoptosis" when
it occurs before the first postirradiation mitosis. Apoptosis, therefore,
occurs rather quickly after irradiation, usually within 4 to 6 hours, and
is abundant in those cell systems in which apoptosis is the primary mode
of cell death.

Cells with the morphologic features of apoptotic cells are also observed
in cultured cell systems in which reproductive cell death is the primary
mode of cell demise. These cells appear much later after irradiation, ie,
24 to 96 hours, and usually with less abundance. Such cells, therefore,
may be displaying the features of apoptosis subsequent to reproductive
cell death. This process will be referred to as "secondary apoptosis."

Loss of Tumor Clonogens--The distinction between apoptosis and
secondary apoptosis is not trivial in the context of radiation response,
for two reasons. First, cells that die by apoptosis directly contribute
to radiation response in appropriate cultured cell systems because, in
those cases, there is usually a one-to-one relationship between an apoptotic
cell and the loss of cells from the clonogenic pool. Thus, a radiation
dose that would induce 50% of the cells to undergo apoptosis would reduce
clonogenic survival to 50%.

Secondary apoptosis does not contribute to radiation response in the
same way. In cultured cell populations in which secondary apoptosis occurs,
cells die primarily by reproductive cell death; this process is quite sufficient,
in and of itself, to remove cells from the clonogenic pool, usually by
inducing a permanent growth arrest, a senescence-like state. There is no
need for apoptosis under these conditions.

Thus, secondary apoptosis probably does not contribute to overall cell
killing and radiation response because it most likely occurs in cells that
have already permanently lost their reproductive capacity regardless of
whether they display the features of apoptosis. In general, there is not
a one-to-one relationship between the proportion of cells undergoing secondary
apoptosis and cell killing because a much higher proportion of the cells
in such a population usually undergo reproductive cell death than display
apoptotic-like features.

Cell Radiosensitivity--Second, and most important, cell populations
for which apoptosis is the primary mode of cell death following irradiation
may be more sensitive to radiation than are cells whose primary mode of
death is reproductive cell death. In such cultured cell systems, substantial
apoptosis is usually induced by doses of 5 Gy or less, and the apoptotic
cells are visualized within a few hours of irradiation. In contrast, secondary
apoptosis generally requires doses higher than 5 Gy and appears more than
24 hours after exposure. Ian Radford clearly established these principles
in a 1991 paper,[10] and they have been verified in subsequent reports.[11,12]

Thus, the observation of cells with the features of apoptosis is not
necessarily indicative of cell radiosensitivity[12] because the relative
sensitivity of the cells in question may depend on whether they are undergoing
apoptosis or secondary apoptosis. It should also be pointed out that radiation-induced
apoptosis appears to be a feature of cultured cells of hematologic or lymphoid
origin, whereas fibroblasts and cultured cells of epithelial origin tend
to undergo reproductive cell death and secondary apoptosis following irradiation.[12]

In Vivo Cell Systems

Compared to what has been accomplished using cultured cell systems,
evaluation of radiation-induced apoptosis in systems irradiated in vivo
has been very limited. Nonetheless, several critical evaluations of apoptosis
in irradiated normal and tumor tissues have revealed important differences
between in vitro and in vivo systems.

Early Studies--Work in this area prior to 1980 was reviewed by
Kerr and Searle.[13] They pointed out that Pratt and Sodicoff[14] reported
the presence of apoptotic bodies in irradiated salivary glands and that
Potten[15] observed apoptosis in the epithelium of intestinal crypts following
irradiation. In addition, Kerr and Searle[13] observed apoptosis in a transplantable
mouse tumor, a sarcoma, that had received radiation.

Thus, this early work established that, in in vivo systems, radiation-induced
apoptosis could occur in cells of nonhematologic origin. Through a meticulous
quantitative analysis of radiation-induced apoptosis in the intestinal
crypt, Potten[15] established two critical points concerning this mechanism
of cell death: (1) Apoptotic cells appeared very quickly following treatment,
with their numbers peaking between 3 and 4 hours after irradiation. (2)
Only some of the cells in the crypt responded to radiation by undergoing
apoptosis, but those that did were exquisitely sensitive to this mode of
cell death; apoptotic figures were detected following radiation doses as
low as .05 Gy.

M. D. Anderson Studies--Since these early reports, systematic
assessments of radiation-induced apoptosis have been carried out in normal
and tumor tissues. Our group's interest in apoptosis was stimulated by
an observation made in the radiation oncology clinic, where irradiation
of the salivary glands is often unavoidable in the treatment of head and
neck cancer. The response of these glands to radiation is remarkable, in
that some patients develop decreased salivary function during the first
week of therapy, having received less than 10 Gy.[16]

To determine the underlying mechanisms responsible for these troublesome
low-dose sequelae, adult female monkeys were irradiated with single radiation
doses of 2.5 to 15 Gy.[17] Damage to the salivary gland was assessed from
sequential biopsy specimens taken 1 to 72 hours after irradiation. The
proportion of apoptotic cells in the tissues was determined by counting
the cells displaying the morphologic features of apoptosis. Apoptotic cells
could be observed as early as 1 hour after irradiation, and most such cells
appeared within 24 hours. Moreover, apoptosis was seen even with the lowest
dose used, 2.5 Gy.

These findings are similar to those observed for cultured cell systems
undergoing apoptosis with respect to dose-responsiveness and kinetics of
appearance. They also suggest that, whereas the salivary gland cells are
terminally differentiated, cells need not be in the cell cycle or even
have reproductive capacity to undergo apoptosis following irradiation.

Heterogeneity of Apoptotic Propensity--Examination of apoptosis
in model tumor systems has led to the appreciation that apoptosis may play
a role in the response of at least some types of tumors to radiation. The
intent of our initial studies[18] was simply to determine whether apoptosis
was a feature of irradiated tumors. Two transplantable murine tumors were
chosen based on their known radiation response: a very radioresistant (TCD50
more than 80 Gy) hepatocarcinoma, HCa-I, and a moderately sensitive (TCD50
= 53 Gy) ovarian adenocarcinoma, OCa-I. (TCD50 refers to the
dose required to cure 50% of the tumors.) These tumors were grown in the
hind legs of mice and were given radiation doses of 25 Gy or more; histologic
sections were prepared from tumors removed at 6, 24, 96, and 144 hours
after treatment.

This first experiment showed that apoptosis occurred in the OCa-I tumor
but not in the HCa-I tumor. Moreover, the apoptotic index was highest at
6 hours following 25 Gy of radiation, declined thereafter, and did not
increase with doses over 25 Gy.

A subsequent, more detailed study[19] pinned down the time course and
dose response for apoptosis induction in the OCa-I tumor. This study showed
that the apoptotic index in the OCa-I tumor peaks at about 30% to 35% of
the cells in the histologic sections between 3 and 4 hours after irradiation
(Figure 1). Doses of 2.5 Gy induced substantial
apoptosis, and the dose response actually leveled off at doses higher than
about 7.5 Gy (Figure 2), suggesting that
only a subset of cells in the tumor have the propensity for radiation-induced
apoptosis. Thus, it became clear from our first two studies that apoptotic
propensity was heterogeneous both among different tumor types and even
among cells within a given tumor.

These instances of heterogeneity were confirmed in a more elaborate
assessment of radiation-induced apoptosis in 14 additional types of murine
tumors.[20] This analysis illustrated that some types of tumors, such as
adenocarcinomas of the mammary gland and ovaries and lymphomas, have an
apoptotic response, whereas other types, such as squamous cell carcinomas,
hepatocarcinomas, and fibrosarcomas, do not. Moreover, for tumors that
displayed an apoptotic response, their dose responses plateaued at an apoptotic
index of 30% at doses higher than 10 Gy.

For many of these tumors, we had previously determined their TCD50
and specific growth delay characteristics in response to single doses of
radiation. Therefore, we produced correlation plots (Figure
3
) of radiation-induced apoptosis vs TCD50 and specific
growth delay for the murine tumors included in the analysis described above.
These plots showed that tumors that responded to radiation with significant
apoptosis tended to have lower TCD50 values (.1 less than P
less than .2) and longer specific growth delays (P less than .05).

For each respective tumor, we also plotted the value for spontaneous
apoptosis measured in the nonirradiated tumor vs the value for radiation-induced
apoptosis (Figure 4). This correlation
was highly significant (P less than .001), suggesting that spontaneous
levels of apoptosis predict the apoptotic response to treatment.

Impact of Dose Fractionation--The studies described above utilized
single doses of radiation, whereas in radiation therapy the dose is given
in fractions. The possible influence of dose fractionation was assessed
using the OCa-I tumor.[21] Two protocols were tested: (1) two doses of
12.5 Gy separated by various intervals up to 5 days and (2) five daily
fractions of 2.5 Gy.

These experiments showed that the protocol using two 12.5-Gy doses produced
a net total proportion of apoptotic cells of about 45% when the two doses
were separated by 5 days (total dose, 25 Gy). The daily 2.5-Gy protocol
produced about 50% net apoptotic cells after 5 days (total dose, 12.5 Gy),
and a single dose of 25 Gy produced only 36% apoptotic cells. Thus, an
apoptotic subpopulation of cells reemerged between doses in the fractionated
protocols, with the daily 2.5-Gy protocol being the most effective.

Role of Apoptosis in Tumor Response

All of the observations discussed thus far are consistent with the possibility
that apoptosis plays a significant role in tumor response to radiation
therapy. However, these are only correlations; cause-and-effect relationships
are more difficult to establish. Moreover, it is hard to envision how apoptotic
indexes of 30% to 35% dictate tumor response in light of a discussion by
Dewey et al,[9] which illustrated that killing this proportion of cells
in a tumor could reduce the TCD50 by only about 4 to 5 Gy. In
contrast, the difference in TCD50 between the sensitive and
resistant tumors used in our analysis was 20 to 30 Gy. Therefore, the correlations
presented above, while intriguing, cannot directly account for the sensitivity
of the tumors analyzed in the study.

One possible explanation is that apoptosis indirectly influences tumor
response. The spontaneous levels of apoptosis scored in the untreated tumors
may be depicting the rates of cell loss in these tumors, and this factor
may, in turn, dictate the number of tumor clonogens, a critical parameter
governing tumor response to radiation. The number of clonogens in these
transplantable tumors can be estimated from the TD50 (a value
representing the number of cells required to yield a tumor in 50% of the
injection sites in a tumor transplantation assay). In an assessment of
the TD50 values for the murine tumors used in our studies,[22]
we confirmed that tumors that have a high spontaneous apoptotic index and
respond to radiation by undergoing apoptosis tend to have a high TD50,
suggesting that their number of tumor clonogens is low compared to those
relatively radioresistant tumors that lack apoptosis as a feature.

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