Recent clinical research of new drug development has focused on a new chemotherapeutic agent: paclitaxel (TAX3), a diterpenoid plant product derived from the bark of the Western yew Taxus brevifolia, which has shown a broad activity in preclinical screening studies and a substantial activity against several refractory tumors in clinical setting (1, 2).

In vitro, this agent appears to have a mechanism of action by promoting microtubule assembly and stabilizing tubulin polymer formation (3) with consequent block in G2-M phase of the cell cycle of proliferating, treated cells (4); its cytotoxicity is time-concentration dependent.

Radiosensitizer effect of TAX has been investigated extensively on the rationale that G2+M is the most radiosensitive phase of the cell cycle (5, 6). In most of these combination studies, a significant radiation potentiating effect of TAX was found only after a TAX given dose that produced both a block in G2-M phase and also a high cell killing (≅90%); in the clinical setting, this could result in damage to normal tissue. Moreover, literature data are contradictory; the combination of prolonged drug exposure (24 h) and XRT resulted in additive, supraadditive, or antagonistic interactions in a wide type of cell lines; the presence of arrest in G2-M did not seem to be a sufficient condition to enhance radiation sensitivity (7, 8). Other mechanisms of TAX-XRT interaction could provide for a synergistic effect.

Therefore, recent works (9, 10) investigated new combination schedules with low TAX doses (subtherapeutic doses) that did not result in cell cycle perturbation to obtain information on: (a) the best synergistic effect as a result of the optimal combination of cell line characteristics, growth phase, drug concentration, and scheduling; and (b) the cellular mechanism of action involved for TAX-mediated radiosensitization as cytokinetic alterations, interference with postirradiation DNA repair, apoptosis, and reoxygenation (11, 12).

The aim of this study was to test different combinations of chemotherapeutic schedules and radiotherapy fractionated to obtain additional information of possible usefulness in the clinical setting.

### Drugs and XRT

TAX was kindly supplied by Bristol-Myers Squibb and was diluted in sterile DMSO to a stock concentration of 1 mg/ml. Working concentrations were diluted in Earle’s balanced salt solution (Life Technologies, Inc., Merelbeke, Belgium) and prepared fresh from the frozen state just before use. Escalating doses ranging from 0.5 to 1,000 nm were used (0.5, 1, 5, 10, 50, 100, and 1,000) for HT29 cells and to 10,000 nm for LoVo cells. The IC50 was calculated for each time exposure. Each sample, with or without concomitant drug exposure, was irradiated in Petri dishes (1-cm thick) at room temperature (22–25°C), using an X-ray unit (Philips: 200 kV, 20 mA, 0.1 mm Cu filter- XK 2600/00) at a dose rate of 0.6 Gy/min. Three schedules of combined TAX-XRT treatment were assayed in HT29 and LoVo cell lines:

### Cell Culture

The human colon adenocarcinoma cell lines, HT29 and LoVo, were provided by Dr. G. Zupi (Laboratory of Experimental Chemotherapy, Regina Elena Institute for Cancer Research, Rome, Italy). HT29 cells were grown in RPMI 1640 supplemented with 10% FCS, 1% l-glutamine, and 0.1% antibiotics; HT29 grew with doubling time of about 19 h. LoVo cells were grown in Nutrient Mixture F-12 with 15% FCS, 1% MEM, bicarbonate, sodium hydroxide, and 0.1% antibiotics; LoVo grew with doubling time of about 30 h. HT29 and LoVo cells were routinely subcultured using a 5-min exposure to Trypsin-EDTA solution and grown at 37°C in a humidified 5% CO2/95% air atmosphere. All products for cell culture were from Life Technologies, Inc.

### Clonogenic Assay

According to the most used and confirmed methods (13), for all experiments stock cultures were washed with EBSS, trypsinized, rinsed, and plated into 60-mm Petri dishes and incubated in fresh medium for 3 days prior to experiments so that the treatment was conducted in the phase for exponential growth. After exposure, cells were washed, trypsinized, harvested as a single cell suspension, and counted with Coulter Counter Model ZM (Coulter Electronics Ltd., Luton Beds, United Kingdom). Known aliquots were dispensed into Petri dishes in triplicate, and colony-forming efficiency was determined from the plating of cells after an incubation period of 2 weeks. Colonies were stained with 2% methylene blue (Carlo Erba, Milano, Italy), and only colonies containing 50 or more cells were counted. Cloning efficiency of the untreated cells (control) was normalized to 100%, and the cloning efficiency of treated cells was expressed as a percentage of control survival.

### Data Analysis

The amount of drug that reduces the proliferation of treated cells of 50% of that controls (IC50) was deduced from a linear regression analysis of dose-response curves. To characterize the radiosensitivity of the cell line and the change in radiosensitivity after treatment with TAX, the MID was calculated. Radiation survival curves were fitted with either one of the linear- quadratic equations:

$$ln\mathit{S}\ {=}\ {-}{\alpha}\mathit{D}\ {-}\ {\beta}\mathit{D}^{2}\ or\ ln(\mathit{S}/\mathit{S}_{TAX})\ {=}\ {-}{\alpha}\mathit{D}\ {-}\ {\beta}\mathit{D}^{2}$$
⁠, were D is the radiation dose, S is the colony survival relative to the plating efficiency, and STAX is the relative survival of unirradiated cells exposed to TAX. According to Fertil et al.(14), coefficient α and β were used to integrate the area under the curve using a 12-point Gaussian formula and to obtain the MID value. SER was calculated by dividing the dose of radiation required to kill 90% of control cells by the dose of radiation required to kill 90% of cells exposed to TAX. The additivity status was determined from the ratio of survival to combined treatment (SCOMB) relative to the product of survival to radiation (SXRT) and drug alone (STAX). SCOMB/SXRT·STAX >1 defines infraadditive interaction; SCOMB/SXRT·STAX <1 indicates supraadditive interaction.

### Flow Cytometry

#### DNA Analysis.

Progression of HT29 and LoVo cells through the cell cycle was determined by flow cytometry. At the end of each treatment, the cells (at least duplicate samples for each point) were washed, harvested, pooled, and then counted. The cell suspension was centrifuged at 1000 rpm for 10 min and fixed with 50% acetone-methanol (1:4 v/v) and 50% PBS at 4°C for at least 1 h before flow cytometric analysis. Approximately 1 × 106 fixed cells were suspended in a volume of 800 μl containing 75 KU/ml RNase (Sigma Chemical Co., St. Louis, MO) and 50 μg/ml PI (Sigma) for 30 min at room temperature. The fluorescence emission of stained cells was measured using a Cytoron Absolute Ortho (Becton Dickinson, San Jose, CA). DNA-dye complex fluorescence was excited by an argon ion laser. Flow studies measured the number of cells versus DNA content and allowed for the determination of the fraction of cells in each phase of cell cycle.

#### BrdUrd Analysis.

DNA synthesis was assessed by incorporation of BrdUrd (Sigma) and by flow cytometric analysis. Cells were incubated with BrdUrd (10 μm) before completion of treatment with TAX and/or XRT. At the end of the pulse labeling period, BrdUrd containing medium was aspirated, and the cells were washed twice with PBS; finally, fresh culture medium was added, and the cultures were further incubated at 37°C. At various intervals of time after BrdUrd labeling (postlabeling times, t = 0, 2, 4, 24, and 48 h) cultures were trypsinized to obtain a monocellular suspension, washed with PBS, and fixed with 70% ethanol. Cells were then washed and treated with HCl 4N for 30 min at 37°C. After washing, the pellets were resuspended in 1 ml of Na2B4O7 (pH 8.5) for 5 min, washed, treated with PBS-Triton X-100 0.5% for 3 min, and incubated with anti-BrdUrd-FITC (Becton Dickinson) for 30 min at 37°C. Finally, the samples were washed twice and resuspended in 1 ml of PBS containing 5 μg/ml of PI and 5.5 μg/ml of RNase for 30 min at 37°C before flow cytometric analysis.

#### Detection of Apoptosis.

Apoptotic cells were detected by flow cytometric analysis using a Cytoron Absolute Ortho. PI was excited using a 488-nm line of an argon ion laser; each sample was analyzed using a very low threshold on the forward scatter signal to include in the analysis all of the populations in the samples. Because relatively early during apoptosis cells detach from the surface of the culture flask and float in the medium, floating cells were added to the trypsinized ones and measured together. Apoptosis was determined by two different flow cytometric techniques using nonpermeabilized and permeabilized cells (15). In the first procedure, nonpermeabilized cells were supravitally exposed to PI to identify apoptotic cells from living and necrotic cells, even in those cases in which low molecular weight DNA fragments do not take place. PI supra-vital exposure leaded to a difference in apoptotic and nonapo-ptotic cells in the ability of excluding the nonvital dye due to a membrane alteration typical of the apoptotic cells; in the second method, permeabilized cells were exposed to PI to determine the hypodiploid peak (sub-G1 peak). The cells were centrifuged at 1000 rpm for 5 min and then processed with two different methods: (a) nonpermeabilized cells (1 × 106) were resuspended in 0.5 ml PBS and then incubated with 1 ml of 40 μg/ml PI solution and 3 μg/ml RNase for 1.5 h at 37°C before the flow cytometric analysis. Apoptotic cells were detected on an forward scatter/PI dot plot as PIdim peak; and (b) 1 × 106 cells were fixed in 2 ml of 70% cold ethanol at 4°C for 30 min. The cells were then centrifuged, washed twice in PBS, and resuspended in 0.5 ml PBS and 1 ml of hypotonic solution containing 40 μg/ml PI, and 3 μg/ml RNase. The mixed cells were incubated in the dark at room temperature for 10 min before the flow cytometric analysis. Analysis of DNA content was measured and displayed using a linear scale, which provided better assurance that objects with minimal DNA content are excluded from the analysis. Twenty thousand events/sample were acquired. As additional controls, a few samples were incubated with 0.1% sodium azide (Sigma), a substance that induces cell death through a nonapoptotic mechanism, and 100 nm dexamethasone (Sigma), a compound with apoptotic activity.

### Analysis of p53, bcl-2, and bax Expression

For all experiments, cells were fixed and permeabilized using 0.25% paraformaldehyde in PBS (15 min at room temperature), followed by 70% cold methanol (60 min at 4°C). After washing in PBS, 1 × 106 cells were incubated (30 min at 4°C) with 10 μl of FITC-conjugated anti-p53 (clone DO-7; Dako, Milano, Italy) or FITC-conjugated anti-bcl-2 (clone 124; Dako); staining of bax was done with an indirect immunofluorescence method using a rabbit polyclonal IgG1 anti-bax protein (Dako) and a secondary antibody, anti-rabbit FITC conjugated. Negative controls were performed replacing anti-p53 and anti-bcl-2 with mouse IgG1 and anti-bax with rabbit IgG1. Moreover, MCF-7 and SK-BR3 cell lines were used as positive controls for bcl-2 staining and as negative and positive controls for p53 analysis, respectively. The fluorescence intensity of 10,000 cells stained with FITC was measured using a Cytoron Absolute Ortho.

### Morphological Analysis of HT29 and LoVo Cells

For morphological assessment, attached and detached cells were counted and washed, and cytocentrifuge slides were prepared, air-dried, and stained by May Grünwald-Giemsa technique. Cell morphology was examined by light microscopy using a Zeiss Axiophot-2 microscope. Cell death was determined based on the expression of morphological features characteristic either of apoptosis (e.g., cell shrinkage, nuclear condensation and fragmentation, extensive formation of membrane blebs, and apoptotic bodies) or of necrosis (e.g., cell swelling, nuclear expansion, and gross cytolysis). The morphology of at least 500 cells was scored from each sample to determine the frequency of apoptotic death.

### Effect of Chronic Exposure to TAX on Cell Survival and Cell Cycle Progression of HT29 and LoVo Cells.

Cell survival was determined by a clonogenic assay. Table 1 shows the SF of exponentially growing HT29 cells as a function of different times of exposure (24–120 h) to escalating concentrations of TAX ranging from 0.5 to 1000 nm. Cells incubated with the same concentration of DMSO were not affected (data not shown). In HT29 cells, survival and cell cycle proliferation during 120 h of exposure (Tables 1 and 2) were not affected by the lowest TAX doses (0.5–1 nm); chronic exposure to 5 and 10 nm TAX induced a decline of cell survival and a moderate and temporary G2-M block. The SF was 69% (5 nm) and 51% (10 nm) after 24 h and gradually recovered to 100 and 80%, respectively, after 120 h. The SF of HT29 cells was greatly reduced only with concentrations of TAX up to 50 nm at all times of exposure (Table 1). Cell cycle analysis at 50 nm TAX showed a broad peak in the lower channel numbers, indicating that cells had lost DNA and were damaged. After 72 h of 50 nm TAX incubation, this peak become more extensive (∼80% of treated cells), including a portion of cells that overlapped the channels for cells with G1-S-phase content (Table 2). Concentrations of drug up to 50 nm also resulted in a stable G2-M block (at all times of incubation). Recovery studies did not show repair of drug damage and cycling out of G2-M block at these high doses (data not show).

The response of LoVo cells to chronic TAX exposure (Table 1) were similar to that of HT29 cells, but LoVo cells were at least 40 times less sensitive after 24 h of TAX exposure; in fact, the mean IC50 was in the range of 10 nm for HT29 cells and 400 nm for LoVo cells. In contrast to the HT29 cells, LoVo cells showed a great enhanced cell killing only after 48 h of TAX exposure (IC50 = 20 nm and IC50 = 8 nm for HT29 and LoVo, respectively). The difference in sensitivity may be due to different doubling times of the two cell lines (19 and 30 h for HT29 and LoVo cells, respectively). After 72 h of TAX exposure, an increase in LoVo cell survival with 1–100 nm TAX was also noticed. Table 3 shows the effect of chronic exposure (24–120 h) to different TAX concentrations on cell cycle distribution of LoVo cells. Below 50 nm TAX, the phase fraction of treated cells did not differ significantly from the control; concentrations up to 50 nm TAX resulted in the characteristic G2-M block at all tested times without production of the nuclear damage found in HT29.

### HT29 and LoVo Cell Survival in Combined Treatment with TAX and XRT.

The first schedule was predominantly additive in HT29 cells and additive or infraadditive in LoVo cells. Flow cytometric analysis did not show perturbation of cells cycle progression in both HT29 and LoVo cells (data not shown).

In the second schedule, the TAX-XRT interaction was additive and infraadditive in HT29 and LoVo cells, respectively. No alteration of cell cycle distribution was found (data not shown).

In the third schedule, the effect of combined treatment was first investigated as a function of the XRT dose using fixed drug doses. TAX doses below the IC50 value were able to increase radiosensitivity of HT29 cells at radiation doses starting from 2 Gy (Fig. 1,A). The rate of radiosensitization was dependent on the concentration of TAX, with the highest decrease in MID value at 10 nm TAX: MID = 2.89 Gy for 3 × 2 Gy alone (combination of three treatment days with 2 Gy XRT) and MID = 1.5 Gy for 10 nm TAX and three times 2 Gy; maximal SER was 2.3 with 10 nm TAX. In contrast in LoVo cells, the TAX-XRT interaction produced a profile similar to that determined for XRT alone (Fig. 1 B), and no significant decrease of MID values was found.

The drug concentration dependence of HT29 and LoVo cell survival with concomitant exposure to radiation is shown in Fig. 2. The dose radiation of 2 Gy was chosen because it represents a clinically relevant radiotherapeutic dose. In HT29 cells, the combination of 3 × 2 Gy in continuous presence of TAX was supraadditive and dose dependent over 0.5 nm TAX. Moreover, posttreatment recovery study showed a loss of cell survival and the inability to overcome the damage in the following 96 h after the end of treatment (Fig. 3,A). These survival data correlated with alteration produced on cell cycle progression compared with control (Table 4). In fact, at the end of treatment, most of the HT29 cells were accumulated overall in S phase, S-G2 junction, and to a smaller extent in the G2-M phase with concomitant depletion of the G1 compartments. This figure persisted after 96 h of posttreatment recovery, indicating that progression through the cell cycle was delayed (1–5 nm TAX) or blocked (10 nm).

Bivariate flow cytometric analysis of the cell cycle progression using pulse-chose BrdUrd labeling showed that at the end of treatment, BrdUrd-positive HT29 cells were decreased in treated cells with respect to control (Fig. 4). Treated BrdUrd-positive cells demonstrated an initial delay at the S-G2 junction, and after 24 h of pulse labeling, the G2-M block and the small G1 repopulation suggest that only a few BrdUrd-positive cells could progress through the block. The duration of the G2-M delay is related to the level of unrepaired damage present in the cells as they approach mitosis (16). Moreover, after treatment, there was a percentage of BrdUrd-negative HT29 cells in S phase that was not able to progress in the following recovery day. Twenty-four h after the pulse labeling, BrdUrd-negative cells initially in the G1 compartment had time to reach this S phase block, but while the cohort of control BrdUrd-negative HT29 cells progressed into the following G1 phase, treated BrdUrd-negative cells remained blocked in S and G2-M phase after 96 h of recovery (data not shown). In HT29 cells, the third schedule seems to produce the inability to overcome DNA damage, as shown by impaired postrecovery; stabilization of the microtubule dynamics and radiation repair could be interrelated. In HT29 cells, treatment with XRT alone (3 × 2 Gy; SF, 65%) induced a smaller accumulation of tumor cells into S and G2-M in comparison to the TAX-XRT combination. The HT29 cells were able to progress through the block and replenished the G1 compartment. In fact, after 24 h of pulse labeling, BrdUrd-positive cells (Fig. 4) were delayed with respect to control but also were able to progress into the following G1 phase after 48 h of recovery (data not shown).

Fig. 2,B shows the effect of drug concentration on fixed radiation doses of 1–2 Gy in LoVo cells. Because of the great reduction of SF obtained with 3 × 2 Gy (SF, 15%), we also chose for combination experiments with the 1-Gy doses (SF, 42%). A weak supradditive interaction was showed only in the presence of 10–50 nm TAX and 3 × 2 Gy; at these doses, the progression through the cell cycle was arrested and was only resolved after 72 h of posttreatment recovery. Interaction between TAX and 3 × 1 Gy was weakly additive and did not produce alteration of cell cycle progression respect to controls (Table 5). Analysis of post-XRT recovery did not produce an appreciable variation of cytotoxicity for up to 96 h (Fig. 3 B).

### Identification of Apoptotic Cells.

Apoptosis is the major mechanism by which ionizing radiation and most chemotherapeutic agents cause tumor cell death (17). By flow cytometric analysis of DNA content, apoptotic cells were quantitatively evaluated by two different methods (see “Materials and Methods”).

Fig. 4 and Table 6 illustrate sub-G1 apoptotic HT29 peak. Very few spontaneous apoptotic HT29 cells were detected in the control (0.6%). After treatment with 3 × 2 Gy alone, the number of apoptotic cells represent 5% of the total cell number. Treatment with TAX alone induced apoptosis only after 24 h of drug-free medium: 0.6% (1 nm), 2% (5 nm), and 8% (10 nm). Higher doses of TAX treatment resulted in DNA fragmentation and G2-M arrest but not in apoptosis. The number of apoptotic HT29 cells detected, following the third schedule of combination, was increased with respect to control and strictly dependent on the TAX concentrations: 9% (1 nm), 13% (5 nm), and 22% (10 nm) at the end of treatment and 6% (1 nm), 8% (5 nm), and 15% (10 nm) after 24 h of drug-free medium. HT29 incubation with sodium azide markedly enhanced the number of dead cells but did not produce evidence of apoptotic cells (0%), whereas dexamethasone induced only a small percentage of apoptotic cells (8%). Similar percentages of apoptotic cells (Table 6) were obtained by analysis of supravital exposure to PI, which was able to give quantitative information about apoptotic cell populations and necrotic cells. No evidence of apoptotic cell death was obtained in LoVo cells (data not shown).

Morphological criteria of identification of apoptotic and necrotic cells were taken into consideration in conjunction with flow cytometric analysis. Microscopic examination of treated HT29 cells (Fig. 5) revealed changes in cell morphology typically seen in cells undergoing apoptosis as cell shrinkage, nuclear condensation, and fragmentation. In particular, HT29 cells treated with 10 nm TAX and 3 × 2 Gy also exhibited formation of “apoptotic bodies,” rupture of the apoptotic cells into debris, and morphology suggestive of necrosis. In contrast, morphological features characteristic of apoptosis were not observed in LoVo cells (data not shown).

### Flow Cytometric Analysis of p53, bcl-2, and bax Expression.

It has long been recognized that apoptotic cell death can be controlled by various stimuli acting as promoters (e.g., bax, Fas, and p53), effectors (interleukin 1β converting enzyme-like protease), or inhibitors (e.g., bcl-2, Mcl-1, and Bcl-XL; Ref. 18). The expression of apoptosis-associated proteins p53, bcl-2, and bax were detected by flow cytometry in both cell lines. With the p53 antibody DO-7, HT29 and LoVo cells were positive and negative, respectively (Table 7). The treated LoVo cells showed an increased of positive p53 (∼30%). After treatments, in HT29 cells no changes in the expression of p53 were observed. This suggests that a p53-independent pathway could be involved in the apoptotic process. bax and bcl-2 proteins are able to form homo- and heterodimers; therefore, it has been proposed that the relative sensitivity of cells to apoptotic stimuli is dependent on the bax:bcl-2 ratio (19, 20). HT29 cells inherently expressed a higher bax:bcl-2 ratio than LoVo cells. This could have contributed to the differences in sensitivity to undergo apoptosis. Moreover, the TAX treatments were able to weakly decrease the expression of antiapoptotic bcl-2 protein in HT29 cells.

The interaction between radiation and chemotherapy may be roughly divided into four generic types: (a) spatial cooperation in which each modality treats different anatomical site; (b) toxicity independence in which the respective toxicities are not overlapping; (c) radioprotection in which normal tissues are protected by noncytotoxic agents; and (d) enhancement of tumor response in which the interaction may be additive, infraadditive, or supraadditive. The in vitro role of TAX as radiosensitizer was widely studied. The obtained results were contradictory and depended on cell lines, drug concentration, and time scheduling of TAX-XRT.

The TAX-XRT interaction was found supraadditive in human astrocytoma (5), breast (6), pancreas (8), ovarian (9, 10), cervical (9, 21, 22), leukemia (23), lung (24), prostate (25), and head and neck (21, 26) carcinoma cell lines and glioma cell lines (27). Other studies have not reported any radiosensitization effect in lung carcinoma (6), colon (28), and cervix cell lines (29). Moreover, some reports showed contradictory results in the same cell line as in prostatic (25, 28), breast (6, 28), and cervical squamous carcinoma (9, 30).

However, on the basis of the above studies (5, 9, 28, 30), it seems that the presence of the G2-M block may not be the main condition for a synergistic interaction between TAX and XRT, but more than one mechanisms may act as TAX concentration, exposure time, time scheduling of drug and XRT, interference with postirradiation DNA repair, p53 expression, apoptosis, and cell line type (9, 10, 19). Moreover, it seems important to obtain a radiosensitization effect to: (a) perform an accurate timing and duration of TAX treatment to allow the use of low TAX concentrations, which did not result in cell cycle perturbations and toxicity; and (b) find the optimal time scheduling of TAX and XRT.

This study investigated the optimal sequence of combination in two human colon adenocarcinoma cell lines (HT29 and LoVo) and was based on previous works indicating that TAX pretreatment at 48 h prior to XRT (9, 10) and/or chronic exposure of low TAX concentration in combination with fractionated irradiations led to a higher radiosensitizing effect (24).

The induction of radiosensitization by TAX was shown to be strongly schedule dependent. In HT29 cells, the first and second schedules resulted in an additive effect, whereas a supraadditive interaction was detected with the third schedule; this effect was obtained with amounts of TAX lower than IC50 and was dose dependent.

In LoVo cells, TAX showed a weak radiosensitization effect. The first and second schedules of combination were infraadditive, whereas the third schedule was additive. Also, an additive interaction between drug and XRT can be considered a good radiosensitization (26). The interaction between XRT and TAX in LoVo cells was dependent on XRT dose but not on TAX concentration, and the additive effect was obtained with XRT dose with a high cell killing and an amount of drug in the range of IC50. HT29 and LoVo cells showed a different sensitivity to drug alone and to radiation alone.

HT29 cells were more sensitive to TAX exposure than LoVo cells. Differential sensitivity was dependent on time of exposure to the drug and was, probably, due to a different proliferation rate; a similar IC50 was obtained after 24 h of TAX treatment in HT29 cells (10 nm; doubling time, 19 h) and after 48 h in LoVo cells (8 nm; doubling time, 30 h).

LoVo cells (MID, 1.27 Gy) appeared more radiosensitive than HT29 cells (MID, 2.89 Gy) and showed a different cell cycle redistribution after XRT exposure. Loss of cellular ability to repair DNA damage and other biochemical and molecular processes can be considered main factors in enhancing radiosensitivity. In contrast to LoVo cells, a wide shoulder characterized the HT29 radiation survival curve, which reflects the accumulation and repair of sublethal lesions. In fact, post-XRT recovery studies of the cytotoxicity showed an enhancement of survival in HT29 cells and an inability to overcome the damage in LoVo cells.

The supraadditive interaction in HT29 cells was obtained with amounts of TAX lower than IC50, which do not result in cell cycle perturbation. These data suggest that a G2-M block is not a condition for TAX to act as radiosensitizer, and more than one mechanism are involved in determining a supraadditive response (9, 10, 26). In fact, the third schedule of combination resulted in a perturbation of cell cycle distribution in an effective impairment of cellular ability in repairing DNA damage and in apoptotic cell death in HT29 cells but not in LoVo cells. Cell cycle distribution (31, 32) and inhibition of postirradiation DNA damage repair (33, 34) are likely to result in supraadditive radiation-drug interaction. The characteristic suppression of the XRT survival curve shoulder in HT29 cells (Fig. 1 B) is likely to proceed from these cooperative effects. Indeed, in LoVo cells, these mechanisms do not share a common pathway, and repair interaction and cytokinetic cooperation appear not to be involved in the combination.

HT29 cells have high constitutional expression levels of p53. LoVo cells are p53 negative, and its expression is induced by treatments. Safran et al.(35) suggest that the response to TAX plus concurrent XRT do not seem to be p53 dependent, and a p53-independent pathway could be activated overall during the combination treatment. The bax:bcl-2 ratio was evaluated in both cell lines; HT29 cells showed a lower ratio than LoVo cells, the value of which is decreased by combined treatment; this could explain the constitutional inability of the LoVo cells to undergo apoptosis. However, HT29 seems to have a low propensity to undergo apoptosis; the absence of p53 wild-type and the bcl-2-positive expression can raise the threshold for apoptosis induction directly or by interfering with the expression of other regulatory apoptotic genes (36, 37).

Our data suggest that the choice of an appropriate time scheduling of drug and radiation permit the use TAX concentrations and radiation doses that may be given in a clinical setting. The radiation-enhancing effect of low TAX doses can be explained by more than one interaction mechanism as the interference with postradiation DNA repair, an impaired cell cycle progression, and apoptosis.

The results of these experiments have great impact on the clinical setting; the treatment of local and/or regional relapses of colorectal cancer is a combination of chemo- and radiotherapy, chiefly in nonpretreated patients. The main clinical goal is to induce tumor shrinkage and to render bulky tumors operable at the time of relapse so that more active radiotherapy is needed with higher cost-benefit for the patients.

If these preclinical data are confirmed in a clinical setting, a realistic benefit for patients will be achieved: a reduction of time exposure to radiation therapy, 3 days of 5 for each week of treatment, a maintenance of the same tumor response with lower radiation dose, and probably reduced toxic local and systemic side effects, which will be able to increase the therapeutic index of a treatment with palliative or radical intent.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by a grant from Istituto Oncologicalo Romagnolo, Forlì, Italy.

3

The abbreviations used are: TAX, Taxol (paclitaxel); XRT, X-ray treatment; IC50, 50% inhibiting concentration; MID, mean inactivation dose; SER, sensitizer enhancement ratio; PI, propidium iodide; BrdUrd, 5-bromo-2′-deoxyuridine; SF, surviving fraction.

Fig. 1.

Radiation survival curves with different TAX concentrations after third schedule administration. A, HT29 cells without (□; MID, 2.89 Gy) or with combined treatment with 1 nm (•; MID, 2.61 Gy), 5 nm (▪; MID, 2.11 Gy), and 10 nm (▴; MID, 1.50 Gy) of TAX. SERs were approximately 1.1, 1.4, and 2.3 for 1, 5, and 10 nm, respectively. B, LoVo cells without (□; MID, 1.27 Gy) or with combined treatment with 5 nm (▪; MID, 1.67 Gy), 10 nm (▴; MID, 1.16 Gy), and 100 nm (; MID, 1.15 Gy) of TAX. SERs were approximately 0.9, 1.2, and 1.5 for 5, 10, and 100 nm, respectively. Data were fitted to the linear quadratic equation given in “Materials and Methods.” Culture medium was changed after 48 h. The effect of TAX alone on cell survival can be seen as the change in survival at 0 Gy. The results are given as the average of three experiments; bars, SD.

Fig. 1.

Radiation survival curves with different TAX concentrations after third schedule administration. A, HT29 cells without (□; MID, 2.89 Gy) or with combined treatment with 1 nm (•; MID, 2.61 Gy), 5 nm (▪; MID, 2.11 Gy), and 10 nm (▴; MID, 1.50 Gy) of TAX. SERs were approximately 1.1, 1.4, and 2.3 for 1, 5, and 10 nm, respectively. B, LoVo cells without (□; MID, 1.27 Gy) or with combined treatment with 5 nm (▪; MID, 1.67 Gy), 10 nm (▴; MID, 1.16 Gy), and 100 nm (; MID, 1.15 Gy) of TAX. SERs were approximately 0.9, 1.2, and 1.5 for 5, 10, and 100 nm, respectively. Data were fitted to the linear quadratic equation given in “Materials and Methods.” Culture medium was changed after 48 h. The effect of TAX alone on cell survival can be seen as the change in survival at 0 Gy. The results are given as the average of three experiments; bars, SD.

Close modal
Fig. 2.

Analysis of the outcome of the third schedule. A, HT29 cells (□, 2 Gy). SF for XRT alone was 65%. B, LoVo cells (▴, 1 Gy; □, 2 Gy). SF for XRT alone was 42 and 15% for 1 and 2 Gy, respectively. The additivity was determined as described in “Materials and Methods.” The results are given as the average of three experiments; bars, SD.

Fig. 2.

Analysis of the outcome of the third schedule. A, HT29 cells (□, 2 Gy). SF for XRT alone was 65%. B, LoVo cells (▴, 1 Gy; □, 2 Gy). SF for XRT alone was 42 and 15% for 1 and 2 Gy, respectively. The additivity was determined as described in “Materials and Methods.” The results are given as the average of three experiments; bars, SD.

Close modal
Fig. 3.

Posttreatment evolution after third schedule administration. A, HT29 cells without (□, 2 Gy) or with combined treatment with 1 nm (•), 5 nm (▪), and 10 nm (▴) of TAX. B, LoVo cells without (□, 2 Gy) or with combined treatment with 5 nm (▪), 10 nm (▴), and 100 nm () of TAX. The results are given as the average of three experiments; bars, SD.

Fig. 3.

Posttreatment evolution after third schedule administration. A, HT29 cells without (□, 2 Gy) or with combined treatment with 1 nm (•), 5 nm (▪), and 10 nm (▴) of TAX. B, LoVo cells without (□, 2 Gy) or with combined treatment with 5 nm (▪), 10 nm (▴), and 100 nm () of TAX. The results are given as the average of three experiments; bars, SD.

Close modal
Fig. 4.

Flow cytometric detection of DNA content, apoptotic cells, and BrdUrd incorporation in HT29 cells. Analysis was performed at the end of the third schedule or TAX-XRT combination (t = 0 h) and after 24 h postrecovery (t = 24 h). Panel 1, control cells; Panel 2, XRT alone ( 3 × 2 Gy); Panels 3–5, cells exposed to the third schedule with 1, 5, and 10 nm TAX, respectively. A, sub-G1 apoptotic peak. Data were obtained from bivariate flow cytometric analysis on DNA fluorescence (PI) or immunofluorescence (BrdUrd). Linear scale was used for analysis of DNA content in fixed cells.

Fig. 4.

Flow cytometric detection of DNA content, apoptotic cells, and BrdUrd incorporation in HT29 cells. Analysis was performed at the end of the third schedule or TAX-XRT combination (t = 0 h) and after 24 h postrecovery (t = 24 h). Panel 1, control cells; Panel 2, XRT alone ( 3 × 2 Gy); Panels 3–5, cells exposed to the third schedule with 1, 5, and 10 nm TAX, respectively. A, sub-G1 apoptotic peak. Data were obtained from bivariate flow cytometric analysis on DNA fluorescence (PI) or immunofluorescence (BrdUrd). Linear scale was used for analysis of DNA content in fixed cells.

Close modal
Fig. 5.

Morphological appearance of HT29 cells treated with the third schedule of combination. A, cells without treatment. B–D, 10 nm TAX + 3 × 2 Gy. The cells were stained with May Grünwald-Giemsa as described in “Materials and Methods.” Treatment induced apoptotic changes such as cell shrinkage, chromatin condensation, nuclear fragmentation, and “apoptotic bodies.” ×500.

Fig. 5.

Morphological appearance of HT29 cells treated with the third schedule of combination. A, cells without treatment. B–D, 10 nm TAX + 3 × 2 Gy. The cells were stained with May Grünwald-Giemsa as described in “Materials and Methods.” Treatment induced apoptotic changes such as cell shrinkage, chromatin condensation, nuclear fragmentation, and “apoptotic bodies.” ×500.

Close modal
Table 1

Survival of HT29 and LoVo cells exposed to different TAX concentrations for various times

Culture medium with TAX was changed after 48 h. Values expressed are percentages of total cells.
Exposure to TAX (h)HT29LoVo
TAX doses (nm)IC50 (nm)TAX doses (nm)IC50 (nm)
0.51510501001,0001510501001,00010,000
24 100a 95 69 51 28 10 100 100 97 67 58 39 400
48 100 91 75 63 22 20 87 61 46 41 35
72 100 100 95 71 21 25 91 73 53 48 37 34
96 100 100 100 78 15 100 97 73 65 42 80
120 100 100 100 80 14 100 100 82 70 48 96
Culture medium with TAX was changed after 48 h. Values expressed are percentages of total cells.
Exposure to TAX (h)HT29LoVo
TAX doses (nm)IC50 (nm)TAX doses (nm)IC50 (nm)
0.51510501001,0001510501001,00010,000
24 100a 95 69 51 28 10 100 100 97 67 58 39 400
48 100 91 75 63 22 20 87 61 46 41 35
72 100 100 95 71 21 25 91 73 53 48 37 34
96 100 100 100 78 15 100 97 73 65 42 80
120 100 100 100 80 14 100 100 82 70 48 96
a

Data represent mean values from three separate experiments; SD <5%.

Table 2

Cell cycle phase distribution of HT29 cells after TAX chronic exposure

Cells were treated with various TAX concentrations and subjected to flow cytometric analysis after treatment as described in “Materials and Methods.” Culture medium with TAX was changed after 48 h.
Treatment%Time (h)
24487296120
G1 55a 66 75 66 72
Control 41 22 18 30 22
G2/M 12
G1 53 66 75 67 71
0.5 nm 42 21 19 27 21
G2/M 13
G1 48 64 62 65 70
1 nm 44 23 25 28 24
G2/M 13 13
G1 44 63 63 63 70
5 nm 40 22 24 27 20
G2/M 16 15 13 10 10
G1 48 63 69 73 71
10 nm 36 22 16 21 21
G2/M 16 15 15
G1 52 34 Fb 52
50 nm 33 52 34
G2/M 15 14 15 14 15
G1 11
100 nm 19
G2/M 95 70 88 90 80
Cells were treated with various TAX concentrations and subjected to flow cytometric analysis after treatment as described in “Materials and Methods.” Culture medium with TAX was changed after 48 h.
Treatment%Time (h)
24487296120
G1 55a 66 75 66 72
Control 41 22 18 30 22
G2/M 12
G1 53 66 75 67 71
0.5 nm 42 21 19 27 21
G2/M 13
G1 48 64 62 65 70
1 nm 44 23 25 28 24
G2/M 13 13
G1 44 63 63 63 70
5 nm 40 22 24 27 20
G2/M 16 15 13 10 10
G1 48 63 69 73 71
10 nm 36 22 16 21 21
G2/M 16 15 15
G1 52 34 Fb 52
50 nm 33 52 34
G2/M 15 14 15 14 15
G1 11
100 nm 19
G2/M 95 70 88 90 80
a

Data represent mean values from three separate experiments; SD, <5%.

b

F, cells or nuclei detected as damaged.

Table 3

Cell cycle phase distribution of LoVo cells after TAX chronic exposure

Cells were treated with various TAX concentrations and subjected to flow cytometric analysis after treatment as described in “Materials and Methods.” Culture medium with TAX was changed after 48 h.
Treatment%Time (h)
24487296120
G1 41a 59 51 63 68
Control 34 16 29 20 17
G2/M 25 25 20 17 15
G1 40 58 56 62 66
1 nm 35 17 24 19 18
G2/M 25 25 20 19 16
G1 40 55 55 61 66
5 nm 35 19 26 17 15
G2/M 25 26 19 22 19
G1 40 51 56 58 68
10 nm 34 22 30 20 12
G2/M 26 27 14 22 20
G1 38 49 51 53 65
50 nm 34 20 21 22 12
G2/M 28 31 28 25 23
G1 37 44 55 44 52
100 nm 30 21 23 25 20
G2/M 33 35 22 31 28
G1 21 18 10
1,000 nm 19 16 35 35 36
G2/M 60 66 55 56 55
G1 14 11 10
10,000 nm 15 11
G2/M 83 71 80 81 85
Cells were treated with various TAX concentrations and subjected to flow cytometric analysis after treatment as described in “Materials and Methods.” Culture medium with TAX was changed after 48 h.
Treatment%Time (h)
24487296120
G1 41a 59 51 63 68
Control 34 16 29 20 17
G2/M 25 25 20 17 15
G1 40 58 56 62 66
1 nm 35 17 24 19 18
G2/M 25 25 20 19 16
G1 40 55 55 61 66
5 nm 35 19 26 17 15
G2/M 25 26 19 22 19
G1 40 51 56 58 68
10 nm 34 22 30 20 12
G2/M 26 27 14 22 20
G1 38 49 51 53 65
50 nm 34 20 21 22 12
G2/M 28 31 28 25 23
G1 37 44 55 44 52
100 nm 30 21 23 25 20
G2/M 33 35 22 31 28
G1 21 18 10
1,000 nm 19 16 35 35 36
G2/M 60 66 55 56 55
G1 14 11 10
10,000 nm 15 11
G2/M 83 71 80 81 85
a

Data represent mean values from three separate experiments; SD, <5%.

Table 4

Analysis of the cell cycle redistribution of HT29 cells after the third schedule of TAX-XRT administration

Cells were exposed to third schedule of TAX-XRT treatment and subjected to flow cytometric analysis after treatment as described in “Materials and Methods.” Culture medium with TAX was changed after 48 h.
TreatmentHours after treatment
024487296
Control
G1 72a 63 73 80 91
S 19 25 16 17
G2/M 12 11
1 nm TAX + 3 × 2 Gy
G1 50 38 37 56 61
S 32 44 46 30 28
G2/M 18 18 17 14 11
5 nm TAX + 3 × 2 Gy
G1 43 34 35 57 59
S 38 48 40 31 30
G2/M 19 18 25 12 11
10 nm TAX + 3 × 2 Gy
G1 43 30 47 49 50
S 41 57 38 39 38
G2/M 16 13 15 12 12
3 × 2 Gy
G1 60 52 61 70 76
S 26 37 27 12
G2/M 14 11 12 18 15
Cells were exposed to third schedule of TAX-XRT treatment and subjected to flow cytometric analysis after treatment as described in “Materials and Methods.” Culture medium with TAX was changed after 48 h.
TreatmentHours after treatment
024487296
Control
G1 72a 63 73 80 91
S 19 25 16 17
G2/M 12 11
1 nm TAX + 3 × 2 Gy
G1 50 38 37 56 61
S 32 44 46 30 28
G2/M 18 18 17 14 11
5 nm TAX + 3 × 2 Gy
G1 43 34 35 57 59
S 38 48 40 31 30
G2/M 19 18 25 12 11
10 nm TAX + 3 × 2 Gy
G1 43 30 47 49 50
S 41 57 38 39 38
G2/M 16 13 15 12 12
3 × 2 Gy
G1 60 52 61 70 76
S 26 37 27 12
G2/M 14 11 12 18 15
a

From three separate experiments; SD, <5%.

Table 5

Analysis of the cell cycle redistribution of LoVo cells after the third schedule of TAX-XRT administration

Cells were exposed to the third schedule of TAX-XRT treatment and subjected to flow cytometric analysis as described in “Materials and Methods.” Culture medium with TAX was changed after 48 h.
TreatmentHours after treatment
0244872
Control
G1 63a 65 69 75
S 20 13 10
G2/M 17 22 24 15
5 nm TAX + 3 × 1 Gy
G1 64 62 71 76
S 19 14
G2/M 17 24 21 15
10 nm TAX + 3 × 1 Gy
G1 66 66 69 75
S 20 13 10
G2/M 14 21 23 15
50 nm TAX + 3 × 1 Gy
G1 65 61 68 74
S 20 14
G2/M 15 25 23 18
3 × 1 Gy
G1 67 61 69 73
S 19 13
G2/M 14 26 25 19
10 nm TAX + 3 × 2 Gy
G1 61 63 60 48
S 16 15 19 26
G2/M 23 22 21 26
50 nm TAX + 3 × 2 Gy
G1 58 60 56 49
S 17 15 20 27
G2/M 25 25 24 24
3 × 2 Gy
G1 60 63 61 65
S 15 15 20 13
G2/M 25 22 19 22
Cells were exposed to the third schedule of TAX-XRT treatment and subjected to flow cytometric analysis as described in “Materials and Methods.” Culture medium with TAX was changed after 48 h.
TreatmentHours after treatment
0244872
Control
G1 63a 65 69 75
S 20 13 10
G2/M 17 22 24 15
5 nm TAX + 3 × 1 Gy
G1 64 62 71 76
S 19 14
G2/M 17 24 21 15
10 nm TAX + 3 × 1 Gy
G1 66 66 69 75
S 20 13 10
G2/M 14 21 23 15
50 nm TAX + 3 × 1 Gy
G1 65 61 68 74
S 20 14
G2/M 15 25 23 18
3 × 1 Gy
G1 67 61 69 73
S 19 13
G2/M 14 26 25 19
10 nm TAX + 3 × 2 Gy
G1 61 63 60 48
S 16 15 19 26
G2/M 23 22 21 26
50 nm TAX + 3 × 2 Gy
G1 58 60 56 49
S 17 15 20 27
G2/M 25 25 24 24
3 × 2 Gy
G1 60 63 61 65
S 15 15 20 13
G2/M 25 22 19 22
a

Data represent mean values from three separate experiments; SD, <5%.

Table 6

Percentage of apoptotic HT29 cells after third schedule administration as identified by three different techniques

TreatmentSub-G1 peakPI supravital exposureMorphology
0 h24 ha0 h24 h0 h24 h
Control 0.6b 0.6 1.6
1 nm TAX 0.6 NDc ND
5 nm TAX ND ND
10 nm TAX 10 ND
3 × 2 Gy
1 nm + 3 × 2 Gy 11 ND ND
5 nm + 3 × 2 Gy 13 12 ND ND
10 nm + 3 × 2 Gy 22 15 20 10 15
Sodium azide, 0.1%
Dexamethasone, 100 nm ND ND ND
TreatmentSub-G1 peakPI supravital exposureMorphology
0 h24 ha0 h24 h0 h24 h
Control 0.6b 0.6 1.6
1 nm TAX 0.6 NDc ND
5 nm TAX ND ND
10 nm TAX 10 ND
3 × 2 Gy
1 nm + 3 × 2 Gy 11 ND ND
5 nm + 3 × 2 Gy 13 12 ND ND
10 nm + 3 × 2 Gy 22 15 20 10 15
Sodium azide, 0.1%
Dexamethasone, 100 nm ND ND ND
a

24 h drug-free medium.

b

Data represent mean values from three separate experiments; SD, <5%.

c

ND, not determined.

Table 7

Percentage of p53, bcl-2, and bax proteins by flow cytometry in HT29 and LoVo cells

Cells were exposed to different TAX-XRT administrations and subjected to flow cytometric analysis as described in “Materials and Methods.”
Cell lines p53 bcl-2 bax
HT29
Control at 4 days of growth 80a 84 89
Control at 7 days of growth 83 86 87
3 × 2 Gy 85 89 88
10 nm TAX for 2 days 82 76 86
10 nm TAX for 4 days 83 75 93
10 nm TAX + 3 × 2 Gy 81 74 98
LoVo
Control at 4 days of growth 96 54
Control at 7 days of growth 98 57
3 × 2 Gy 37 97 63
10 nm TAX for 2 days 30 95 60
10 nm TAX for 4 days 33 94 58
10 nm TAX + 3 × 2 Gy 28 91 61
Cells were exposed to different TAX-XRT administrations and subjected to flow cytometric analysis as described in “Materials and Methods.”
Cell lines p53 bcl-2 bax
HT29
Control at 4 days of growth 80a 84 89
Control at 7 days of growth 83 86 87
3 × 2 Gy 85 89 88
10 nm TAX for 2 days 82 76 86
10 nm TAX for 4 days 83 75 93
10 nm TAX + 3 × 2 Gy 81 74 98
LoVo
Control at 4 days of growth 96 54
Control at 7 days of growth 98 57
3 × 2 Gy 37 97 63
10 nm TAX for 2 days 30 95 60
10 nm TAX for 4 days 33 94 58
10 nm TAX + 3 × 2 Gy 28 91 61
a

Data represent mean values from three separate experiments; SD, <5%.

We thank Dr. G. Zupi of Laboratory of Experimental Chemotherapy, Regina Elena Institute for Cancer Research, Rome, for scientific and technical assistance.

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