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Introduction

The use of preoperative radiation is becoming a widely used form of adjuvant therapy. Radiation often changes a tumor from fixed and unresectable to mobile and resectable by decreasing its size. Patients with a better chance of benefiting from radiotherapy are selected on the basis of size, mobility, differentiation, and stage of the tumor. However, tumors are not equally sensitive to radiation.^1-3

Many times during our practice, we have seen tumors that melted away following radiotherapy, tumors that remained untouched, and tumors that showed a mixture of heterogeneous components (some sensitive to the radiation, and others less so). The importance of knowing in advance which tumor will or will not respond to radiotherapy is obvious. A radiosensitive tumor may require less radiation and thus involve fewer side effects for the patient. On the other hand, a patient with a tumor that is radioresistant may be treated with an alternative form of therapy. Determining the criteria capable of predicting tumor radiosensitivity has attracted both clinicians and basic researchers but with little success. So far, no relationship has been found between a tumor’s clinical features and its sensitivity to radiation.

With the development of newer molecular biology techniques and with the growing knowledge about the regulation of the cell cycle, several attempts are being made to identify molecular markers capable of predicting radiation sensitivity of tumors. This paper provides an overview of the molecular events taking place during radiation-induced cell death and of the attempts to use molecular markers of these events as predictors of radiation sensitivity.

Radiation-Induced Cell Death

Okada et al⁴ described two types of radiation-induced cell death: reproductive and interphase. Reproductive cell death occurs after one or more divisions (it involves actively proliferating cells). This process seems related to radiation-induced chromosomal aberrations with partial inactivation or loss of genetic material. This type of cell death usually occurs following high doses of irradiation and involves cell cycle alterations. Conversely, interphase cell death occurs before the next cell division (it is specific for cells that are not proliferating at the time of the irradiation). With respect to interphase death, two types of cells are identified: sensitive cells (ie, lymphocytes) usually killed by 10 to 100 rads, and resistant cells (ie, hepatocytes) capable of surviving several thousand rads. This type of cell death usually involves the apoptotic pathway.⁴ Therefore, a cell carrying DNA damage as a result of exposure to ionizing radiation either will undergo growth arrest, which can be irreversible or reversible after the DNA has been repaired, or will die by apoptosis. Choosing between these possible responses depends on cell type, location, environment, oncogenes expression, and extent of damage.

Reproductive Cell Death: Cell Cycle Alterations in Irradiated Cells

Checkpoints

Cells do not progress into the next phase of the cell cycle before completing the previous phase. They also have mechanisms to detect any genomic alteration and to stop the progression of the cell cycle once DNA damage is detected. These mechanisms, called checkpoints, are able to arrest cells in the G₁ phase, to slow down cells in the S phase, and to arrest cells in the G₂ phase. At the same time, repair genes are activated. The detection of single-stranded DNA seems to be the signal able to activate the checkpoint mechanisms.⁵

G₁ Checkpoint: Cell-Cycle Regulator p53 — Several years ago, Little⁶ observed that fibroblasts exposed to ionizing radiation were spending longer time in the G₁ phase of the cell cycle. It later became evident that this phenomenon was due to a temporary arrest in the G₁ phase of irradiation-damaged cells, induced by p53.⁷ This is a tumor suppressor gene present on the short arm of chromosome 17, and it is capable of binding as tetramer to specific DNA sequences, acting as a transcription activator.^8,9 The gene p53 is involved in suppressing the proliferation of cells carrying abnormal DNA. It is now known that exposure to radiation induces increased levels of p53 protein. This increase is transient and correlates with the presence of DNA damage. Once the DNA has been repaired, the level of p53 protein returns to normal.¹⁰ Metabolite deprivation, physical damage, heat shock, hypoxia, and oncogene expression can also activate p53.¹¹ The cellular response to the activation of p53 involves the growth arrest of cells in the early stages of the cell cycle (G₁ and G₂); p53 acts as a transcriptional activator inducing the expression of p21/WAF1, a cyclin-dependent kinase (cdk) inhibitor.^8,9 Interaction of this protein with cdk2/cyclin E complex will inhibit the progression of the affected cell to the S phase of the cell cycle (Fig 1).¹²

Fig 1. — Schematic representation of the interactions among the major cell-cycle regulators. When hypophosphorylated, the retinoblastoma protein (Rb) binds and inactivates the transcription factors (TF). TF, when unbound, induce synthesis of proteins involved in cell proliferation. Retinoblastoma protein releases the TF when phosphorylated (P) by cyclins. On the other hand, cyclins are inactivated by p21, a protein under the regulatory control of p53. MDM2, a 90-kD zinc-finger protein, is a negative regulator of p53.

Other genes activated by either p53 or radiation damage include GADD45 and MDM2. GADD45 arrests the cell cycle in the S phase.¹³ MDM2 is involved in the feedback regulation of p53 transcriptional activity, acting during the recovery stage from p53-induced G₁ arrest. MDM2 binds and inactivates p53.¹⁴ p53 can also bind proteins involved in DNA replication (replication protein A), preventing cells carrying damaged DNA from entering the S phase.¹⁵ Dysfunctional (mutated) p53 is expected to allow increased genomic instability and tolerance to DNA damage with consequent increase in cell survival after radiation exposure (Fig 2A-B).

Fig 2A-B. — (A) Cascade of events following the exposure of a cell with functional p53 to radiation. Note the pivotal role of p53 in arresting the cell cycle and activating genes involved in DNA repair. If this mission fails, cells will undergo apoptosis. (B) In cells carrying a nonfunctional p53, cell-cycle arrest and DNA repair cannot occur. This will increase genomic instability and will allow tumor growth despite the exposure to radiation.

S-Phase Checkpoint: the ATM Gene — Initiation and elongation stages of DNA replication are also inhibited by ionizing radiation.¹⁶ It seems that both cis-acting and trans-acting regulatory processes are involved in the inhibition of DNA replication after radiation.^17,18

Interestingly, individuals affected by ataxia telangiectasia (AT), an autosomal recessive disorder, are unable to slow down DNA replication after exposure to radiation. AT cells fail to inhibit both initiation and elongation stages of DNA replication, suggesting the lack of a factor that, in normal cells, delays replication. The AT gene (ATM) regulates DNA replication in irradiated cells.¹⁹ It codes for a protein kinase with homology to the catalytic domain of phosphatidylinositol 3-kinase (PI-3 kinase). This protein is constitutively expressed during the cell cycle and serves as a checkpoint gene in response to DNA damage. It appears that ATM may regulate p53-mediated apoptosis (Fig 3A-B).²⁰

Fig 3A-B. — The ataxia telangiectasia gene (ATM). (A) In patients with AT, the ATM gene is mutated and cannot downregulate DNA replication in response to radiation. ATM is also an activator of p53. (B) In non-AT patients, wild-type ATM (wATM) induces posttranslational activation of p53 through the phosphorylation of serine residue 15 (ser15P). This change stabilizes p53, preventing it from interacting with its negative regulator MDM2.

It is evident that oncogenes (H-ras, v-myc)²¹ and cell receptors (IGF1-R)²² may also inhibit DNA replication in irradiated cells. p53 is not involved in the S-phase checkpoint.²³

G₂ Phase Checkpoint — Postsynthetic cycling cells exposed to radiation are arrested in the G₂ phase.²⁴ This G₂ delay seems to be caused by either a reduction in the level of cyclin B or by a delay in the activation of cdks normally acting during this phase of the cell cycle. Therefore, it is not surprising that both of these alterations occur in irradiated cells.^25,26 In addition, since the activation of cdks relies on the dephosphorylation of cdk1 by cdc25, lack in activation of cdc25 will also result in a prolonged G₂ phase (Fig 4). A reduction in cdc25 phosphorylation has been described in irradiated cells.²⁷

Fig 4. — Cell cycle and cyclins. Cyclin D, D1-3, and E are synthesized and are active in the G1 phase. They regulate the transition from the G1 to the S phase. To do so, they must be phosphorylated (P). Cyclins A and B1-2 control the transition from G2 to M. To exercise this action, cyclin B must bind to cdc2, a constitutively produced inactive kinase. The complex cyclin B/cdc2 is activated by kinases and phosphatases (cdc25) inducing mitosis (M). Following mitosis, the cells can reenter the cell cycle or pass to a quiescent state (G0).

Interphase-type Radiation-Induced Cell Death: Programmed Cell Death

Apoptosis

Apoptosis (programmed cell death) is the process by which a cell is able to trigger its own death. Cells able of undergoing apoptosis usually have specific cell-death receptors. The best characterized death receptor is CD95 (Fas or APO-1). The Fas receptor and its ligand (FasL) are components of the tumor necrosis factor gene superfamily. The binding of FasL to FasR induces trimerization of the receptor with activation of the death-inducing signaling cascade (caspases). This ultimately stimulates the death-effector molecule interleukin-1beta-converting enzyme (ICE) that, when activated, induces apoptotic cell death in hours (Fig 5).^28,29 It seems that cells carrying deregulated genes are primed for apoptosis. Eventually, additional mutations occurring during tumor progression will disable the apoptotic response, thereby facilitating uncontrolled tumor expansion.

Fig 5. — Some of the major steps involved in the Fas-induced death-signaling pathway.

Recent studies indicate that several cell-cycle regulators may also be critical players in the apoptotic response. The requirement of a functional p53 for apoptosis to occur is demonstrated by the fact that mouse embryo fibroblasts derived from p53 knockout mice are refractory to myc-induced apoptosis.¹¹ It has become evident that p53 is also capable of modulating apoptosis by suppressing Bcl-2 (through the activation of Bax),³⁰ by downregulating the anti-apoptotic receptor insulin-like growth factor 1 (IGF1-R),³¹ and by inducing the expression of binding protein IGF-BP3³² and proteins regulating angiogenesis.³³ Transrepression of antiapoptotic genes has also been postulated.³⁴ Rb is another suppressor of apoptosis. It seems that caspase-dependent degradation of Rb is essential for Fas-induced CD95, and drug-induced apoptosis to occur.³⁵ Furthermore, it is becoming evident that after DNA damage, the Fas receptor and its ligand are activated through a p53-dependent mechanism.³⁶

To explain similar associations, Harrington et al³⁷ proposed a dual signal theory in which activation of cell proliferation will prime the cellular apoptotic program that, unless shut down by survival signals, will automatically remove the affected cells. According to this theory, the balance between the proapoptotic and antiapoptotic signals will determine whether a cell will proliferate or die. This view implies the participation of other factors such as Bcl-2. When overexpressed, this protein suppresses apoptosis and slows down the cell cycle.³⁸ Conversely, the proapoptotic Bax protein accelerates cell cycle progression and antagonizes Bcl-2.³⁹ It is interesting that one way p53 can modulate apoptosis is through the activation of Bax and the suppression of Bcl-2.³⁰

This already intricate network of signals, connecting pathways with disparate and sometimes contrasting functions, becomes even more complex as new discoveries are made. For example, Ashkenazi et al⁴⁰ recently described decoy receptors (DcR1 and DcR2) capable of preventing Apo2L from binding to and activating death receptors DR4 and DR5.

Molecular Markers of Radiation Sensitivity

It is possible that any of the factors involved in the regulation of cell cycle and/or apoptosis may represent a marker of tumor sensitivity to radiation. For example, recent studies have shown that, independently of the cell type, the presence of mutated p53 usually predicts tumor resistance to radiation.^15,41-43 McIlwrath et al⁴⁴ and Hamada et al⁴⁵ have shown that cells carrying p53 mutations are more resistant to radiation and chemotherapy than are cells with functional p53. However, there is some disagreement among authors about this correlation.^46,47 Fu et al⁴⁸ recently reported that 95.5% of colorectal tumors with p53-positive and p21-negative staining (using immunohistochemistry) were radioresistant, while 83.3% of p53-negative tumors and p21-positive tumors were radiosensitive. Preliminary studies at our institute also suggest that a positive reaction to p53 is usually associated with radioresistant tumors. The positivity for p53 implies a mutated p53 with prolonged half-life that can be detected by immunohistochemistry (Fig 6A-B).

Fig 6A-B. — (A) A radioresistant, invasive, colonic adenocarcinoma shows intense and diffuse p53 immunostain, indicating the presence of a mutated P53 protein. Note the nuclear selective localization of the stain. Overlying normal colonic mucosa is p53-negative. (B) In contrast, a later-proven radiosensitive tumor shows negative p53 immunostaining. The negativity of the stain indicates the presence of a functional p53.

Tumor radiosensitivity has also been associated with inhibition of the erbB receptors,⁴⁹ with the use of monoclonal antibody to the epidermal growth factor receptor,⁵⁰ and with the activation of p34cdc2 kinase.⁵¹ Du-145 prostate cancer cells have one deleted and one truncated Rb gene and are resistant to radiation-induced apoptosis. Bowen et al⁵² showed that reintroduction of Rb in these cells was associated with their increased sensitivity to radiation-induced apoptosis. On the same line, Sakakura and colleagues⁵³ reported that overexpression of Bax enhances the radiation sensitivity in breast cancer cells.

Conclusions

Radiation is a well-established cancer treatment modality. However, it is limited by the toxicity it produces in the adjacent normal tissues. Therefore, a challenge for the radiation oncologist is to decrease the damage to the normal tissue while applying sufficient doses of radiation to destroy the tumor tissue. Dissection into the molecular mechanisms following the irradiation of cells opens a new avenue to antitumor strategy. A few reports have recently appeared in the literature describing tumor cell manipulations that can reverse radioresistant phenotypes to radiosensitive phenotypes.^54-56 If confirmed, this strategy would allow targeting of tumor cells using minimal doses of radiation. Preclinical applications of these strategies are underway.

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