Ingela Turesson

p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2-stimulated angiogenesis

The p38 mitogen–activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor–dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2–induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2–induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2–induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2–induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2–driven angiogenesis.

The relationship between cellular radiation sensitivity and tissue response may provide the basis for individualising radiotherapy schedules

There is a wide variation in normal tissue reactions to radiotherapy and in many situations the severity of these reactions limits radiotherapy dose. Clinical fractionation studies carried out in Gothenburg have demonstrated that a large part of the spectrum of normal tissue reactions is due to differences in individual normal tissue sensitivity. If this variation in normal tissue reactions is due to differences in intrinsic cellular radiosensitivity, it should be possible to predict tissue response based on measurement of cellular sensitivity. Here we report the initial results of a study aimed at establishing whether a direct relationship exists between cellular radiosensitivity and tissue response. Ten fibroblasts strains, including four duplicates, were established from a group of patients in the Gothenburg fractionation trials who had received radiotherapy following mastectomy. Skin doses were measured and both acute and late skin changes were observed following radiotherapy. Right and left parasternal areas were treated with different dose fractionation schedules. Clonogenic assays were used to assess intrinsic cellular radiosensitivity, and all experiments were carried out without prior knowledge of the clinical response, or which strains were duplicates. Irradiation was carried out using 60Co gamma-rays at high dose-rate (HDR) of 1-2 Gy/min and low dose-rate (LDR) of 1 cGy/min. A spectrum of sensitivity was seen, with SF2 values of 0.17-0.28 at HDR and 0.25-0.34 at LDR, and values of D0.01 of 5.07-6.38 Gy at HDR and 6.43-8.12 Gy at LDR. Comparison of the in vitro results with the clinical normal tissue effects shows a correlation between cellular sensitivity and late tissue reactions, which is highly significant with p = 0.02. A correlation between cellular sensitivity and acute effects was noted in the left-sided parasternal fields, but not the right. This is thought to be coincidental, and without biological significance. Our results suggest that cellular sensitivity might form the basis for the development of an assay system capable of predicting late normal tissue effects to curative radiotherapy, which might allow dose escalation in some patients. Increased local control and cure, with unchanged or improved normal tissue complications, could result from such individualised radiotherapy prescriptions.

DESCRIBING PATIENTS' NORMAL TISSUE REACTIONS: CONCERNING THE POSSIBILITY OF INDIVIDUALISING RADIOTHERAPY DOSE PRESCRIPTIONS BASED ON POTENTIAL PREDICTIVE ASSAYS OF NORMAL TISSUE RADIOSENSITIVITY

Clinical radiotherapeutic doses are limited by the tolerance of normal tissues. Patients given a standard treatment exhibit a range of normal tissue reactions, and a better understanding of this individual variation might allow for individualisation of radiotherapeutic prescriptions, with consequent improvement in the therapeutic ratio. At present, there is no simple way to describe normal tissue reactions, which hampers communication between clinic and laboratory and between groups from different centres. There is also no method for comparing the severity of reactions in different normal tissues. This arises largely because there is no definition of a “normal” reaction, an “extreme” reaction or the particular term “over‐reactor” (OR). This report proposes definitions for these terms, as well as a simple terminology for describing normal tissue reactions in patients having radiotherapy. The “normal” range represents the individual variation in normal tissue reactions amongst large numbers of patients treated in the same way which is within clinically acceptable limits. The term “OR” is applied to an individual whose reaction is more severe than the normal range but also implies that this forced a major change in the radiotherapeutic prescription or that the reactions were very severe or fatal. A “severe OR” would develop serious problems with a typical radical dose, while an “extreme OR” would have such difficulties at a much lower dose. To describe the normal range, a numerical scale is suggested, from 1 to 5, resistant to sensitive. The term “highly radiosensitive” (HR) is suggested for category 5. An “informal” relative scale, as suggested here, is quick and simple. It should allow comparison between different hospitals, compensate for differences in radiotherapeutic dose and technique and allow comparison of reactions between different anatomical sites. It should be adequate for discriminating patients at the extremes of the normal range from those at the centre. It is hoped that the definitions and terminology proposed here will aid communication in the field of predictive testing of normal tissue radiosensitivity. Int. J. Cancer (Pred. Oncol.) 79:606–613, 1998.

Biological response to radiation therapy.

In an investigation by the Swedish Cancer Society, the present status, critical issues and future aspects and potentials were described by an expert group for each of nine major areas of radiation therapy research. This article deals with biological response to radiation. Separate sections deal with molecular responses to radiation, the stem cell and clonogenic cell concepts and the importance of cell proliferation, cell and tissue responses to doses above and below 1 Gy, respectively, the potential role of intercellular signalling pathways, the so-called bystander effect and radiation biology-based therapy planning and treatment optimization.

Low-dose hypersensitive γH2AX response and infrequent apoptosis in epidermis from radiotherapy patients

BACKGROUND AND PURPOSEnA low-dose hypersensitivity to radiation can be observed in vitro for many human cell types in terms of increased cell kill per dose unit for doses below 0.5Gy. Quantification of the double-strand break marker gammaH2AX in samples taken in clinical radiotherapy practice has the potential to provide important information about how induction and repair of severe DNA damage and apoptosis are linked to low-dose hypersensitivity.nnnMATERIAL AND METHODSnThe effects of exposure to low doses (0.05-1.1Gy) were investigated in skin biopsies taken from prostate cancer patients undergoing the first week of radiotherapy. gammaH2AX foci and apoptotic cells were visualised by immunohistochemistry and quantified by image analysis.nnnRESULTSnThe gammaH2AX foci pattern in biopsies taken 30min after a single fraction revealed a low-dose hypersensitivity below 0.3Gy (p=0.0009). The result was consistent for repeated fractions (p=0.00001). No decrease in foci numbers could be detected when comparing biopsies taken 30min and 2h after single fractions of 0.4 and 1.2Gy. The result was consistent for repeated fractions. Only 43 of 168,000 cells in total were identified as apoptotic, yet a dose dependency could be detected after 1week of radiotherapy (p=0.003).nnnCONCLUSIONSnWe describe a method based on gammaH2AX to study DNA damage response and apoptosis in a clinical setting. A gammaH2AX hypersensitive response to low doses can be observed in epidermal skin, already 30min following delivered fraction. A very low frequency of apoptosis in normal epithelium suggests that this effect is not an important part of the in vivo response to low doses.

A low-dose hypersensitive keratinocyte loss in response to fractionated radiotherapy is associated with growth arrest and apoptosis

BACKGROUND AND PURPOSEnThe existence of a hypersensitive radiation response to doses below 0.5Gy is well established for many normal and tumour cell lines. There is also evidence for hypersensitive tissue responses in acute skin damage and kidney function in mice. Recently, we have identified that a hypersensitive gammaH2AX response exists in human epidermis. The aim of this study was to investigate the dose-response of basal clonogenic keratinocytes in normal skin to fractionated radiotherapy with low dose fractions.nnnMATERIALSnSkin punch biopsies were taken before and during radiotherapy from prostate cancer patients undergoing radiotherapy with a curative intent. Areas of epidermis receiving daily fractions of approximately 0.1, 0.2, 0.45 and 1.1Gy were biopsied on the same occasion to determine dose-response for each individual patient. In total, 89 cases were assessed either at 1, 2.5, 3, 4, 5 or 6.5 weeks in the treatment course. Biopsy sampling of another 25 patients was performed from areas receiving 0.45 and 1.1Gy per fraction at regular intervals throughout the 7-week treatment period. The number of basal keratinocytes per mm of the interfollicular epidermis was determined. The DNA damage response of the basal keratinocytes was investigated by immunohistochemical staining for molecular markers of growth arrest, mitosis and cell death, using p21, phospho-H3 and gammaH2AX, respectively. The number of stained keratinocytes in the basal layer was counted manually. The p21 staining was also quantified by digital image analysis.nnnRESULTSnThe individual dose-response relationships revealed a low-dose hypersensitivity for reduction of basal keratinocytes throughout 7 weeks of radiotherapy (p<0.01). Growth arrest and cell proliferation assessed at 1 week and 6.5 weeks showed, in both cases, hypersensitive increase of p21 (p<0.01) and hypersensitive depression of mitosis (p<0.01). Manual counting and digital image analysis of p21 showed good agreement. Cell death was infrequent but increased significantly between 1 and 6.5 weeks and displayed a hypersensitive dose-response at the end of the treatment period.nnnCONCLUSIONSnA low-dose hypersensitivity in basal skin keratinocyte reduction is present throughout 7 weeks of radiotherapy. A persistent hypersensitive growth arrest response and cell killing mediate this effect.

Comparison of DNA repair protein expression and activities between human fibroblast cell lines with different radiosensitivities.

In order to investigate the molecular basis of variation in response to ionising radiation (IR) in radiotherapy patients, we have studied the expression of several genes involved in DNA double‐strand break repair pathways in fibroblast cell lines. Ten lines were established from skin biopsies of cancer patients with different normal‐tissue reactions to IR, and 3 from a control individual. For all 10 test cell lines, the cellular radiosensitivity was also known. Using Western blots we measured, in non‐irradiated cells, the basal expression levels of ATM, Rad1 and Hus1, involved in the control of cellular DNA damage checkpoints, together with DNA‐PKcs, Ku70, Ku80; XRCC4, ligaseIV and Rad51, involved in radiation‐ induced DSB repair. We also analysed the in vitro enzymatic activities, under non‐irradiated conditions, of the DNA‐PK and XRCC4/ligaseIV complexes. The levels of expression of the different proteins were similar in all the cell lines, but the activities of the DNA‐PK and XRCC4/ligaseIV complexes showed some differences. These differences did not correlate with either the normal tissue response of the patient in vivo or with cellular radiation sensitivity in vitro. The activity differences of these enzyme complexes, therefore, do not account for the variation of responses seen between patients. Int. J. Cancer 85:845–849, 2000.

Effects of a dietary intervention on acute gastrointestinal side effects and other aspects of health-related quality of life : A randomized controlled trial in prostate cancer patients undergoing radiotherapy

PURPOSEnTo study the effect of a dietary intervention on acute gastrointestinal side effects and other aspects of health-related quality of life (HRQOL) in prostate cancer patients referred to radiotherapy.nnnMATERIALS AND METHODSnA total of 130 patients were randomly assigned to one of two groups: an intervention group (IG, n=64), instructed to reduce their intake of insoluble dietary fibres and lactose, a standard care group (SC, n=66), instructed to continue their normal diet. Gastrointestinal side effects and other aspects of HRQOL were evaluated from baseline up to 2 months after completed radiotherapy, using the EORTC QLQ-C30 and QLQ-PR25 and the study-specific Gastrointestinal Side Effects Questionnaire (GISEQ). A scale indicating adherence to dietary instructions was developed from a Food Frequency Questionnaire (FFQ), with lower scores representing better compliance. Descriptive and inferential statistical analyses were conducted.nnnRESULTSnThere was an interaction effect between randomization and time in the FFQ Scores (p<0.001), indicating that both groups followed their assigned dietary instructions. The dietary intervention had no effect on gastrointestinal side effects or other aspects of HRQOL. During radiotherapy, the percentage of patients with bowel symptoms and bloated abdomen was lower in IG compared to SC, but the between-group differences were not statistically significant. During radiotherapy, the percentage of patients with bowel symptoms, urinary symptoms, pain, fatigue and diminished physical and role functioning increased in both groups.nnnCONCLUSIONSnThe dietary intervention had no effect on gastrointestinal side effects or other aspects of HRQOL. The tendency towards lower prevalence of bowel symptoms in IG may indicate some positive effect of the dietary intervention, but methodological refinements, clearer results and longer follow-up are needed before the value of diet change can be established with certainty.

Folate-metabolizing genes in lymphoma patients from Sweden.

To the Editor: Non-Hodgkin lymphomas are a very heterogeneous group of tumours and mortality varies greatly depending on sub-type. The most common types of NonHodgkin lymphomas are follicular lymphoma (FL) and diffuse large B-Cell lymphoma (DLBCL) which together comprise 60–65% of all Non-Hodgkin lymphomas [1, 2]. However, much effort has been made in the investigation of the risk of developing Non-Hodgkin lymphomas, FL and DLBCL in relation to the presence of certain polymorphisms, particularly polymorphisms in folatemetabolizing genes such as TYMS, MTHFR, MTR and SHMT1 [3–11]. Folate-metabolizing genes are involved in the DNA synthesis and methylation of DNA, which if incorrectly expressed because of polymorphism or mutation could lead to cancer development [10]. Numerous reports were subsequently published where the authors had found significant differences in genotype frequencies when Non-Hodgkin lymphomas, FL and DLBCL were compared with normal controls. However, the results of these analyses were often inconsistent between the studies [3–11]. These reports encouraged us to investigate a Swedish cohort of lymphoma patients and controls for the most evaluated polymorphisms in the folate metabolic pathway (TYMS 3R or 2R, TYMS IVS6–68C > T, TYMS 1122A > G, MTHFR 677C > T, MTHFR 1298 A > C, SHMT1 1420C > T and MTR 2776 A > G). Herein, we present genotypes from 260–277 patients with NHL,

Effects of a dietary intervention on gastrointestinal symptoms after prostate cancer radiotherapy: Long-term results from a randomized controlled trial

BACKGROUND AND PURPOSEnTo evaluate the long-term effects of dietary intervention on gastrointestinal symptoms after highly dose-escalated radiotherapy for localized prostate cancer, using boost with protons or high-dose-rate brachytherapy.nnnMATERIALS AND METHODSnPatients were randomized to an intervention group (n=64) advised to reduce insoluble dietary fiber and lactose intake, or to a standard care group (n=66) advised to continue their usual diet. Gastrointestinal symptoms, other domains of health-related quality of life (HRQOL), and dietary intake were evaluated for ⩽24months post-radiotherapy with the European Organization for Research and Treatment of Cancer quality-of-life questionnaires QLQ-C30 and QLQ-PR25, Gastrointestinal Side Effects Questionnaire, and Food Frequency Questionnaire. The effect of the intervention on gastrointestinal symptoms was evaluated using generalized estimating equations.nnnRESULTSnDietary intervention had no obvious effect on long-term gastrointestinal symptoms or HRQOL. The intervention group markedly reduced their dietary fiber and lactose intake during radiotherapy, but adherence tended to decline over time. The vast majority of long-term gastrointestinal symptoms were reported as a little, with a noticeable difference from pre-treatment only for unintentional stool leakage, limitations on daily activities, and mucus discharge.nnnCONCLUSIONnLong-term gastrointestinal symptoms were predominantly mild, and dietary intervention was not superior to a usual diet in preventing these symptoms.