Intravenous injection of bortezomib, melphalan and dexamethasone in refractory and relapsed multiple myeloma.

BACKGROUND: A combination of bortezomib (1.3 mg/m(2)), melphalan (5 mg/m(2)), and dexamethasone (40 mg) (BMD), with all three drugs given as a contemporary intravenous administration, was retrospectively evaluated. PATIENTS AND METHODS: Fifty previously treated (median 2 previous lines) patients with myeloma (33 relapsed and 17 refractory) were assessed. The first 19 patients were treated with a twice-a-week (days 1, 4, 8, 11, 'base' schedule) administration while, in the remaining 31 patients, the three drugs were administered once a week (days 1, 8, 15, 22, 'weekly' schedule). RESULTS: Side-effects were predictable and manageable, with prominent haematological toxicity, and a better toxic profile in 'weekly' schedule (36% versus 66% in 'base' schedule). The overall response rate was 62%. After median follow-up of 24.5 months (range 2.7-50 months), the median progression-free survival (PFS) was 21.6 with no difference between the two schedules and the median overall survival (OS) was 33.8 months. Independently from the adopted schedule, we found that also in a cohort of relapsed/refractory patients achieving at least partial remission improved PFS (35.2 versus 9 months) and OS (unreached median versus 18 months). CONCLUSION: Taken together, our observations suggest that BMD is an effective regimen in advanced myeloma patients with acceptable toxicity.

All-trans retinoic acid converts E2F into a transcriptional suppressor and inhibits the growth of normal human bronchial epithelial cells through a retinoic acid receptor- dependent signaling pathway.

Retinoids, including retinol and retinoic acid derivatives, maintain the normal growth and differentiation of human bronchial epithelial (HBE) cells and are under investigation as agents for lung cancer prevention. In this study, we examined the biologic effects of retinoids on normal HBE cells and the molecular mechanisms of retinoid actions. At a dose of 10(-6) M, all-trans retinoic acid (t-RA) suppressed the proliferation of normal HBE cells, which accumulated in the G0 phase. No evidence of programmed cell death was observed. The class of retinoid nuclear receptor that mediated the growth arrest was explored. Normal HBE cell growth was suppressed by a retinoid that selectively activates retinoic acid receptors but not by one that activates retinoid X receptors. The E2F transcription factor has demonstrated a role in G0 entry through transcriptional suppression of genes that induce cell cycle progression. To investigate the role of E2F in retinoid signaling, transient transfection assays were performed using reporter plasmids containing E2F-binding sites. Findings from these experiments suggested that t-RA treatment converted E2F into a transcriptional suppressor. Supporting this possibility, t-RA inhibited the expression of the E2F target genes B-myb, cyclin A, and cyclin E. Further, t-RA increased the levels of nuclear E2F-4, p107, and p130 and enhanced the binding of E2F-4 to p107, which have been associated with the conversion of E2F into a transcriptional suppressor in other cells. These findings point to retinoic acid receptor- and E2F-dependent pathways as potential mediators of retinoid-induced growth arrest in normal HBE cells and have implications for the use of retinoids in clinical trials on the prevention of lung cancer.

Activation of p21WAF1/Cip1 expression by a temperature-sensitive mutant of human p53 does not lead to apoptosis.

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 (p21) inhibits cellular proliferation and has been implicated in p53-dependent apoptosis. In this study, we examined the regulation of p21 in the K562 erythroleukemia cell line using a human temperature-sensitive mutant of p53, 143Ala. We showed that 143Ala at the permissive temperature (32 degrees C) activated the expression of p21. Stable cell lines expressing 143Ala or other p53 mutants were established. We then examined whether elevation of p21 promotes apoptosis and sensitized cells to radiation-induced apoptosis. Our results showed that p21 elevation did not promote or sensitize K562 cells to apoptosis.

Differential p53 phosphorylation and activation of apoptosis-promoting genes Bax and Fas/APO-1 by irradiation and ara-C treatment.

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation- and ara-C-induced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-C-treated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.

CD30 ligand in lymphoma patients with CD30+ tumors.

PURPOSE: CD30 ligand (CD30L), which is expressed on resting B and activated T lymphocytes, can induce cell death in several CD30+ cell lines. Patients with CD30+ tumors (Hodgkin's disease and Ki-1+ non-Hodgkin's lymphoma) frequently have elevated soluble CD30 (sCD30) levels in their serum, which correlates with a poor prognosis. The role of sCD30 in protecting tumor cells from CD30L-mediated cell death and the pattern of CD30L expression on human peripheral-blood lymphocytes (PBLs) of normal donors and patients with CD30+ tumors are investigated. MATERIALS AND METHODS: CD30L surface protein expression was determined by two-color flow cytometry on PBLs of patients with CD30+ tumors and normal individuals. CD30L levels were determined on subsets of PBLs before and after stimulation with phytohemagglutinin (PHA), anti-CD3 antibody, or CD40L. sCD30 was measured by enzyme-linked immunosorbent assay (ELISA). The apoptotic activity of membrane-bound CD30L was tested in a CD30+ cell line by the annexin V-binding method. RESULTS: Unstimulated T lymphocytes of normal donors and patients with lymphoma rarely expressed CD30L surface protein, but were able to express it after stimulation with PHA or anti-CD3 antibody. Resting B cells of patients with CD30+ tumors had lower levels of detectable surface CD30L compared with normal donors (mean, 55% and 80.6%, respectively; P = .0008). Patients with high levels of serum sCD30 had lower detectable levels of CD30L on their PBLs (R2 = .72, P = .0008) and exogenous sCD30 blocked membrane-bound CD30L-mediated apoptosis in a CD30+ cell line. CONCLUSION: In patients with CD30+ tumors, sCD30 can decrease the availability of CD30L on PBLs. Blocking the apoptosis-inducing activity of CD30L by its soluble receptor may explain how CD30+ tumors escape immunosurveillance and may be related to the reported poor prognosis of patients who have elevated sCD30 levels.

Phase I study of mitoxantrone plus etoposide with multidrug blockade by SDZ PSC-833 in relapsed or refractory acute myelogenous leukemia.

PURPOSE: Expression of the multidrug resistance gene (MDR1) p170 protein is frequent in leukemic blasts from patients with relapsed acute myelogenous leukemia (AML). A phase I study using the nonimmunosuppressive MDR1 blocker SDZ PSC-833 (PSC) in combination with mitoxantrone (MITO) and etoposide (VP) was performed. PATIENTS AND METHODS: Starting doses (LVL0) of MITO (3.25 mg/m2/d on days 1 and 3 to 6) and VP (210 mg/m2/d on days 1 and 3 to 5) were 40% of the maximal-tolerated dose (MTD) from a prior study. A 1.5-mg/kg loading dose of PSC was followed by a 120-hour continuous infusion of 10 mg/kg/d on days 2 to 6. Blood samples for PSC, MITO, and VP pharmacokinetics (PK) were taken on days 1 and 3, and samples for MDR1 expression were taken on day 0. RESULTS: Severe mucositis developed in all patients at LVL0; therefore, MITO and VP doses were reduced to 2.5 and 170 mg/m2 (LVL-1) for the next seven patients, and this dose proved to be MTD. All LVL0 and three LVL-1 patients had transient elevations in the serum bilirubin level to > or = 4 mg/dL. Serum creatinine level increased to greater than 2 mg/dL in one case. There were no other grade 3 or 4 nonhematologic toxicities observed. The peripheral blood was cleared of leukemia in three LVL0 and four LVL-1 patients. The marrow was cleared of leukemic cells in one LVL0 and five LVL-1 patients, and a significant reduction in marrow leukemic infiltrate was observed in eight of 10. No patient achieved complete remission (CR), and all died of progressive disease (n = 8) or infection (n = 2). MDR1 expression was detected by fluorescent-activated cell sorter (FACS) analysis in five of seven cases. An elevated MDR1 mRNA level was detected by quantitative polymerase chain reaction (Q-PCR) in six of eight cases studied. Clearing of leukemia cells from the marrow occurred in four of six MDR1-positive and one of three MDR1-negative patients. Despite the fact that LVL0 doses had to be reduced due to toxicity, coadministration of PSC did not produce a consistent effect on MITO PK; however, it did repeatedly lead to increased levels of VP in the serum. CONCLUSION: We conclude that PSC-MITO-VP is a tolerable regimen with antileukemic activity. Addition of PSC necessitated a 66% reduction in MITO and VP doses from a prior study without PSC.

Cellular pharmacology of mitoxantrone in p-glycoprotein-positive and -negative human myeloid leukemic cell lines.

Previous reports suggest that resistance to mitoxantrone in different tumor cell lines is unrelated to the overexpression of p-glycoprotein. In order to determine the role of p-glycoprotein in the cellular pharmacology of mitoxantrone flow cytometry and confocal microscopy were used to study two human myeloid leukemia cell lines selected for resistance to mitoxantrone (HL-60MX2) and doxorubicin (HL-60DOX). To optimize the detection of intracellular mitoxantrone, we determined the maximum excitation (607 nm) and emission (684 nm) wavelength by fluorescence spectroscopy. The modified flow cytometric conditions using 568.2 nm laser emission for excitation and a 620 nm long pass filter for fluorescence collection resulted in a 1-log increase in sensitivity, compared with standard 488-nm laser excitation. Uptake and retention of mitoxantrone in the presence of verapamil, a calcium channel blocker known to inhibit p-glycoprotein, were analyzed. Our results showed no change in uptake and retention of the drug in p-glycoprotein-negative mitoxantrone-resistant HL-60MX2 cells and in its sensitive parental line, HL-60s. In contrast, 3.1- and 2.4-fold increases were found in uptake and retention of mitoxantrone in p-glycoprotein-positive cells (HL-60DOX) incubated with verapamil. Confocal microscopy of intracellular drug distribution demonstrated reduced nuclear uptake, which could be reversed by verapamil, in HL-60DOX. A characteristic punctate pattern was observed for the intracytoplasmic drug distribution in HL-60DOX and HL-60MX2 cells and was partially modified by the presence of verapamil in HL-60DOX cells. Verapamil increased cytotoxicity of mitoxantrone two-fold in HL-60DOX cells, 1.4-fold in HL-60MX2, and had no effect in HL-60s. Our study demonstrates that the cellular pharmacology of mitoxantrone is affected by p-glycoprotein and can be reversed at least in part by verapamil. Other mechanisms of resistance however, seem to play a determinant role in the modulation of mitoxantrone cytotoxicity.

2-Chlorodeoxyadenosine therapy for idiopathic hypereosinophilic syndrome.

Idiopathic hypereosinophilic syndrome (HES) is a rare disease with no established effective therapy. It has been reported that interleukin-5 (IL-5) produced by helper T cells plays a major role in the proliferation of eosinophils. The nucleotide analogue 2-chlorodeoxyadenosine (2-CdA), which induces excellent clinical responses in hairy cell leukemia, is known to suppress helper T cells; therefore, we used 2-CdA, alone or in combination with cytarabine, to treat patients with idiopathic HES. 2-CdA alone and combined with cytarabine resulted in a rapid and sustained decrease in circulating eosinophils in two patients with idiopathic HES that was refractory to steroids, hydroxyurea and cytarabine. The efficacy of 2-CdA alone and combined with cytarabine exceeded by far that of cytarabine alone. However, reverse transcription-polymerase chain reaction (RT-PCR) did not show production of IL-5 or granulocyte-macrophage colony-stimulating factor mRNA in T cells as previously reported, and multiple cytokine receptors were found on eosinophils in idiopathic HES, suggesting that IL-5 may not be the sole cytokine involved in the regulation of idiopathic HES. The clinical efficacy of 2-CdA in idiopathic HES needs to be established on a large group of patients.

Fludarabine-mediated inhibition of nucleotide excision repair induces apoptosis in quiescent human lymphocytes.

Incorporation of fludarabine, 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A), into replicating DNA inhibits further chain elongation and is the critical event in F-ara-A-mediated cytotoxicity. We have used the normal cellular process of nucleotide excision repair to create an opportunity for F-ara-A incorporation into the DNA of noncycling cells. Irradiation of quiescent lymphocytes with UV light (254 nm, 0. 5-30 J/m2) in the presence of [3H]F-ara-A produced a dose-dependent increase in F-ara-A monophosphate incorporation into DNA that reflected a 60-70% inhibition of DNA repair at 2 h. Lymphocytes pretreated with 3 micrometer F-ara-A for 2 h before irradiation with 0.5 or 2.0 J/m2 were incubated for 24 h in the presence or absence of F-ara-A. Morphological features of apoptosis and DNA cleavage into high molecular weight fragments were increased in cells treated with UV plus F-ara-A compared to those treated with UV or F-ara-A alone. FACScan analysis confirmed the morphological and biochemical results. A 2-h pulse of F-ara-A produced intracellular F-ara-ATP levels of 40 micrometer, and removal of F-ara-A from the media resulted in a monophasic elimination (r2 = 0.88) in F-ara-ATP levels with a half-life of 5.6 h. Lymphocytes undergoing apoptosis demonstrated a G0 DNA content, indicating that entry into the cell cycle was not required. This study demonstrates that F-ara-AMP is incorporated into DNA during UV-induced repair in quiescent lymphocytes and that this is associated with the inhibition of ongoing DNA repair and an increased incidence of apoptosis. Combinational therapies involving fludarabine with agents and modalities that initiate DNA repair may have clinical relevance in the treatment of human malignancies.

PML/RARalpha, a fusion protein in acute promyelocytic leukemia, prevents growth factor withdrawal-induced apoptosis in TF-1 cells.

A unique mRNA produced by the t(15;17) (q22-24;q11-21) translocation in the leukemic cells of acute promyelocytic leukemia patients encodes a chimeric protein, PML/RARalpha, which is formed by the fusion of the retinoic acid receptor alpha (RARalpha) and the promyelocytic locus gene (PML). This translocation is often the only visible karyotypic aberration present which is detected in almost 100% of acute promyelocytic leukemia patients. As an initial step to study the role of PML/RARalpha in leukemogenesis, we attempted to express the fusion protein in hematopoietic cells through retrovirus-mediated gene transfer of the retroviral vector, pGPRCHT, which contains the PML/RARalpha cDNA. Transduction of the PML/RARalpha cDNA fragment used in this vector, which extends from the position 31 bp to the position 2638 bp in a transcription unit driven by the Moloney murine sarcoma virus LTR, was found to abrogate the growth factor dependence of TF-1 cells. In addition, introduction of PML/RARalpha into TF-1 cells can protect these cells from the apoptosis usually induced in TF-1 cells by growth factor withdrawal, as measured by three assays for apoptosis: morphology, DNA ladder formation, and end labeling of nicked DNA with fluorescent-conjugated nucleotide precursors followed by a fluorescence-activated cell sorting assay. These data suggest that the PML/RARalpha fusion protein may inhibit programmed cell death in myeloid cells.

Mitogenic effect of nerve growth factor (NGF) in LNCaP prostate adenocarcinoma cells: role of the high- and low-affinity NGF receptors.

We have investigated the effect of nerve growth factor (NGF) in the androgen-dependent, prostate adenocarcinoma LNCaP cell line. Exposure of LNCaP cells to NGF resulted in a significant increase of cell proliferation. The effect was concentration dependent and equally present in serum- or charcoal-stripped serum-supplemented and serum-deprived conditions. The mitogenic action of NGF was accompanied by an enhanced expression of prostate-specific antigen (PSA) and resulted additive to the proliferative effect of dihydrotestosterone. The proliferative effect of NGF appeared to be mediated by the high-affinity NGF receptor, p140trka. Only p140trka, but not the low-affinity NGF receptor, p75LNGFR, was expressed in LNCaP cells; both the proliferative response and the phosphorylation of p140trka upon NGF treatment were prevented by the tyrosine kinase inhibitor K252a. LNCaP cells transiently transfected with the cDNA encoding for p75LNGFR appeared more sensitive to NGF, as demonstrated by the increased number of p75LNGFR-transfected LNCaP cells exposed for 72 h to NGF compared with wild LNCaP cultures. However, p75LNGFR-transfected LNCaP cells rapidly underwent apoptotic death when deprived of NGF. Our study demonstrates the physiological relevance of NGF in the regulation of prostate cell proliferation and the relative contribution of the high- and low-affinity NGF receptors in this control.

Inhibition of lung cancer proliferation by antisense cyclin D.

The growth and tumorigenicity of murine lung cancer cells transfected with an antisense cyclin D1 construct were evaluated in studies pertaining to mouse lung carcinogenesis. This antisense construct inhibited the expression of cyclin D in these cells, significantly reducing both their in vitro proliferation and tumorigenicity in nude mice relative to control cells. These data may have implications regarding the treatment of human neoplasms of aerodigestive tract origin that either overexpress the cyclin D oncogene or exhibit mutations that influence cell cycle progression via cyclin D-dependent mechanisms.

Semi-automated PCR method for quantitating MDR1 expression.

The MDR1 gene is involved in drug resistance in many hematopoietic and solid tumors. The Quantitative PCR System 5000 (QPCR-5000; Perkin-Elmer) is a new instrument system that uses electrochemiluminescence to automatically quantitate polymerase chain reaction (PCR) products. A comparative study between radioactively labeled PCR (32P-PCR) and QPCR was performed to analyze the MDR1 gene expression in the drug-resistant (Doxorubicin) cell lines Dox40, Dox6, the parental cell line 8226/S, CEM Dox1 and three acute myeloid leukemia (AML) patient samples. Using the Dox40 and Dox6 resistant cell lines, we compared the sensitivities of QPCR and 32P-PCR. A strong signal was obtained from QPCR at 20 to 25 cycles (which is in the linear range for quantitation), while a weak signal was obtained using 32P-PCR at the same cycle number. Dilution experiments gave better precision with the QPCR than with the radioactive method. AML samples were studied with the MDR1-specific MAbs MRK16 and 4E3, and the efflux function was analyzed using Rh-123 retention in the absence or presence of verapamil. The three samples showed high (D = 0.79), medium (D = 0.52) and negative (D = 0.08) p-glycoprotein (P-gp) levels and correlated with efflux function. The MDR1/beta 2-M mRNA ratios for 32P-PCR were 0.41, 0.40 and 0.12, respectively, and were 0.127, 0.097 and 0.028, respectively, for QPCR. There were significant differences between the samples with high and medium P-gp levels comparing the two methods. Very low levels of MDR1 in CEM Dox1 cells could be detected only by QPCR. In conclusion, QPCR was found to be more reproducible, accurate and sensitive than 32P-PCR.

Enhanced percentage of CD5+ B lymphocytes in newly diagnosed IDDM patients.

The percentage of CD5+ B lymphocytes, the prevalence of islet cell antibodies (ICA) and of anti-insulin autoantibodies (IAA) and HLA-A-B-C and DR antigens were studied in 32 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients, in 12 non-insulin-dependent diabetes mellitus (NIDDM) patients and in 12 healthy subjects. The percentage of CD5+ B lymphocytes ranged from 18% to 51.2% (mean 40.3 +/- 11%) in IDDM patients, whereas in NIDDM patients and in controls it ranged from 20% to 25.2%, (mean 21.3 +/- 4.1%) and from 16% to 24%, (mean 19.3 +/- 1.9%), respectively (P less than 0.01 vs. NIDDM patients and vs. controls). There was no correlation between a higher percentage of CD5+ B lymphocytes and the presence of ICA and/or IAA, and their titres, and/or of any HLA-A-B-C and DR antigens. Thus, an enhanced percentage of CD5+ B lymphocytes may be present in newly diagnosed IDDM patients; the possible role of this cell type in the pathogenesis of IDDM needs further investigation.

Enhanced percentage of Leu M3+DR+ and Leu M3+CD25+ cells in newly diagnosed IDDM patients.

The percentage of Leu M3+DR+ and of Leu M3+CD25+ cells was determined by means of immunofluorescence analysis in a group of patients with insulin dependent diabetes mellitus (IDDM). Our results show that an increased percentage of these cells may occur in the early stage of the disease. These data provide evidence for a "phenotypical" activation of Leu M3+ cells at the onset of the disease and warrant future studies to evaluate the potential role of these cells in the pathogenesis of IDDM.

G-CSF alone vs cyclophosphamide plus G-CSF in PBPC mobilization of patients with lymphoma: results depend on degree of previous pretreatment.

We performed a randomized study to compare 'G-CSF alone' (administered at dose of 10 mcg/kg/day) and 'cyclophosphamide plus G-CSF' (cyclophosphamide at dose of 4 g/m(2) and G-CSF at dose of 10 microg/kg/day), as PBPC mobilization schedules in 52 patients with NHL or HD. Randomization was stratified according to the amount of previous chemotherapy (< or =2 and >2 lines of previous chemotherapy). Mean CD34+ cell peak in P.B., mean 'Total CD34+ cells' harvested and percentage of patients successfully mobilized, in the group mobilized with 'G-CSF alone' vs the group mobilized with 'cyclophosphamide plus G-CSF', were: 35.3 x 10(6) vs 45.8 x 10(6)/l (P=0.3), 5.4 x 10(6) vs 6.8 x 10(6)/kg (P>0.9) and 50 vs 61% (P=0.4). No differences were observed in the stratum of less pretreated patients. However, in the stratum of patients who had previously received more than two lines of chemotherapy, CD34+cell peak (P=0.05) and percentage of successful mobilization (P=0.01) were higher when 'cyclophosphamide plus G-CSF' was used. Using logistic regression, both age and mobilization with 'G-CSF alone' were significantly associated with a low CD34+ cell peak in P.B. However, in the stratum of less pretreated patients, only age was significantly associated with this risk.

Enhancement of apoptosis in irradiated glioblastoma cells by pentoxifylline.

The p53 gene is frequently mutated in human glioblastomas, which are highly resistant to radiation and chemotherapy. Pentoxifylline has recently been shown to sensitize breast cancer cells in which the wild-type p53 function was abrogated to cisplatin-induced apoptosis. In this study, the effect of pentoxifylline on three irradiated human glioblastoma cell lines, U87MG, which has the wild-type p53; U251, which has a mutant p53; and LN-Z308, which has no p53, was examined. After radiation, even at a dose as high as 20 Gy, typical apoptotic cells were rarely produced. G(2) cell cycle arrest was observed with all three cell lines. Significant G(1) cell population was only observed with U87MG cells, in which increased levels of p21/WAF1/Cip1 occurred. Pentoxifylline increased the amount of radiation-induced DNA fragmentation and apoptosis in p53-defective U251 and LN-Z308 cells. Colony formation assay demonstrated that pentoxifylline moderately enhanced the cell killing effect of irradiation in these cells.

Cellular and molecular effects of protons: apoptosis induction and potential implications for cancer therapy.

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.