Rupert Schmidt-Ullrich

Stress and radiation-induced activation of multiple intracellular signaling pathways.

Abstract Dent, P., Yacoub, A., Contessa, J., Caron, R., Amorino, G., Valerie, K., Hagan, M. P., Grant, S. and Schmidt-Ullrich, R. Stress and Radiation-Induced Activation of Multiple Intracellular Signaling Pathways. Radiat. Res. 159, 283–300 (2003). Exposure of cells to a variety of stresses induces compensatory activations of multiple intracellular signaling pathways. These activations can play critical roles in controlling cell survival and repopulation effects in a stress-specific and cell type-dependent manner. Some stress-induced signaling pathways are those normally activated by mitogens such as the EGFR/RAS/PI3K-MAPK pathway. Other pathways activated by stresses such as ionizing radiation include those downstream of death receptors, including pro-caspases and the transcription factor NFKB. This review will attempt to describe some of the complex network of signals induced by ionizing radiation and other cellular stresses in animal cells, with particular attention to signaling by growth factor and death receptors. This includes radiation-induced signaling via the EGFR and IGFI-R to the PI3K, MAPK, JNK, and p38 pathways as well as FAS-R and TNF-R signaling to pro-caspases and NFKB. The roles of autocrine ligands in the responses of cells and bystander cells to radiation and cellular stresses will also be discussed. Based on the data currently available, it appears that radiation can simultaneously activate multiple signaling pathways in cells. Reactive oxygen and nitrogen species may play an important role in this process by inhibiting protein tyrosine phosphatase activity. The ability of radiation to activate signaling pathways may depend on the expression of growth factor receptors, autocrine factors, RAS mutation, and PTEN expression. In other words, just because pathway X is activated by radiation in one cell type does not mean that pathway X will be activated in a different cell type. Radiation-induced signaling through growth factor receptors such as the EGFR may provide radioprotective signals through multiple downstream pathways. In some cell types, enhanced basal signaling by proto-oncogenes such as RAS may provide a radioprotective signal. In many cell types, this may be through PI3K, in others potentially by NFKB or MAPK. Receptor signaling is often dependent on autocrine factors, and synthesis of autocrine factors will have an impact on the amount of radiation-induced pathway activity. For example, cells expressing TGFα and HB-EGF will generate protection primarily through EGFR. Heregulin and neuregulins will generate protective signals through ERBB4/ERBB3. The impact on radiation-induced signaling of other autocrine and paracrine ligands such as TGFβ and interleukin 6 is likely to be as complicated as described above for the ERBB receptors.

Radiation-induced proliferation of the human A431 squamous carcinoma cells is dependent on EGFR tyrosine phosphorylation

Accelerated cellular repopulation has been described as a response of tumors to fractionated irradiation in both normal tissue and tumor systems. To identify the mechanisms by which cells enhance their proliferative rate in response to clinically used doses of ionizing radiation (IR) we have studied human mammary and squamous carcinoma cells which are autocrine growth regulated by the epidermal growth factor receptor (EGFR) and its ligands, transforming growth factor-α and EGF. Both EGF and IR induced EGFR autophosphorylation, comparable levels of phospholipase Cγ activation as measured by inositol-1,4,5-triphosphate production, and as a consequence oscillations in cytosolic [Ca2+]. Activities of Raf-1 and mitogen-activated protein kinase (MAPK) were also stimulated by EGF and IR by Ca2+-dependent mechanisms. All these responses to EGF and IR were dependent upon activation of EGFR as judged by the use of the specific inhibitor of EGFR autophosphorylation, tyrphostin AG1478. Importantly, IR-induced proliferation of A431 cells was also inhibited by AG1478. This is the first report which demonstrates a link between IR-induced activation of proliferative signal transduction pathways and enhanced proliferation. We propose that accelerated repopulation of tumors whose growth is regulated by EGFR is initiated by an IR-induced EGFR activation mechanism that mimics the effects of growth factors.

Factors influencing cosmetic outcome and complication risk after conservative surgery and radiotherapy for early-stage breast carcinoma.

PURPOSE The study was undertaken to assess the relationship among cosmesis and complications to factors related to disease presentation, surgical and radiotherapeutic technique, and adjuvant systemic therapy in conservative treatment for early-stage breast carcinoma. PATIENTS AND METHODS Between 1982 and 1988, 234 women with stage I/II breast carcinoma were treated with conservation therapy by a highly standardized protocol of limited excision and radiotherapy. Radiation boost and/or reexcision were determined by careful quantitation of the normal tissue margin around the primary tumor. Boosts to 20 Gy were preferentially performed with interstitial iridium-192 (192Ir) implants. Axillary node dissections were performed in all patients aged less than 70 years. Adjuvant therapy consisted of cyclophosphamide, methotrexate, (doxorubicin), and fluorouracil (CM[A]F) six to eight times for node-positive premenopausal women and tamoxifen for node-positive or -negative postmenopausal women. Median follow-up was 50 months (range, 20 to 80 months). Cosmesis was graded by defined criteria, and complications were individually scored. RESULTS Factors found to impact cosmesis adversely were palpable tumors (P = .046), volume of breast tissue resected (P = .027), reexcision of the tumor bed (P = .01), number of radiation fields (P = .03), radiation boost (P = .01), and chest wall separation (P = .01). There was a trend toward worse cosmesis (P = .062) in patients receiving tamoxifen. Cosmesis was not adversely affected by interstitial implant in spite of a higher prescribed dose. Factors influencing complication risk were axillary node dissection (P = .02), number of lymph nodes harvested (P = .05), and chemotherapy (P = .03). CONCLUSIONS Optimal cosmesis and minimal complication risk require careful attention to the technical details of surgery and radiotherapy. The impact of systemic therapies needs to be more thoroughly examined.

Signal transduction and cellular radiation responses.

Abstract Schmidt-Ullrich, R.K., Dent, P., Grant S., Mikkelsen, R.B. and Valerie, K. Signal Transduction and Cellular Radiation Responses. Exposure of cells to ionizing radiation results in complex cellular responses resulting in cell death and altered proliferation states. The underlying cytotoxic, cytoprotective and cellular stress responses to radiation are mediated by existing signaling pathways, activation of which may be amplified by intrinsic cellular radical production systems. These signaling responses include the activation of plasma membrane receptors, the stimulation of cytoplasmic protein kinases, transcriptional activation, and altered cell cycle regulation. From the data presented, there is increasing evidence for the functional links between cellular signal transduction responses and DNA damage recognition and repair, cell survival, or cell death through apoptosis or reproductive mechanisms.

Partial breast brachytherapy after lumpectomy: Low-dose-rate and high-dose-rate experience

PURPOSE The use of partial breast brachytherapy (PBB) after lumpectomy for selected patients with early-stage breast cancer reduces the adjuvant radiotherapy treatment time to <1 week. Despite the advantages of accelerated treatment, maintaining an acceptable cosmetic outcome is important. In a cohort of patients who received low-dose-rate (LDR) or high-dose-rate (HDR) PBB after lumpectomy, the clinical characteristics and treatment parameters were analyzed to identify predictors for an unfavorable cosmetic outcome. METHODS AND MATERIALS Early-stage breast cancer patients with clear resection margins and 0-3 positive lymph nodes were eligible for PBB. Uniform guidelines for target definition and brachytherapy catheter placement were applied. The HDR PBB dose was 34 Gy in 10 fractions within 5 days, and the LDR dose was 45 Gy given at a rate of 50 cGy/h. The end points included incidence of radiation recall reaction, telangiectasias, and cosmetic-altering fibrosis. RESULTS Between 1995 and 2000, 44 patients with early-stage breast cancer received PBB without adjuvant external beam radiotherapy after lumpectomy (31 HDR PBB, 13 LDR PBB). After a median follow-up of 42 months (range 18-86), all patients remained locally controlled. The overall rate of good/excellent cosmetic outcome was 79.6% overall and 90% with HDR PBB. Radiation recall reactions occurred in 43% of patients (6 of 14) who received adriamycin. LDR PBB and adriamycin were significant predictors for late unfavorable cosmetic changes in univariate analysis (p = 0.003 and p = 0.005, respectively). CONCLUSION Although a high rate of local control and good/excellent cosmetic outcome is provided with HDR PBB, the risk of unfavorable cosmetic changes when treated with both LDR PBB and adriamycin is noteworthy. This suggests that HDR PBB is preferred in patients for whom adriamycin is indicated.

Postoperative radiotherapy in head and neck carcinoma with extracapsular lymph node extension and/or positive resection margins: A comparative study

In head and neck carcinoma, the finding of extracapsular lymph node extension and/or positive resection margins portends poor locoregional control and survival. The effectiveness of postoperative radiotherapy in these patients has been controversial due to insufficient studies comparing resected patients with those also receiving radiation. Between 1982 and 1988, 441 radical head and neck resections were performed at the Medical College of Virginia. Pathologic review of these cases identified 125 with extracapsular lymph node extension and/or positive resection margins. Of these, 43 had extracapsular lymph node extension only, 24 had both positive resection margins and extracapsular lymph node extension, and 58 demonstrated positive resection margins only. Surgery alone was performed in 71 of these patients while 54 cases received surgery and postoperative radiotherapy, (combined modality treatment) CMT. Radiotherapy doses ranged from 50 to 70 Gy. The surgery alone and combined modality treatment groups were comparable with respect to the distribution of positive resection margins and extracapsular lymph node extension. Slightly more CMT patients had clinical T4 disease compared with the surgery alone group (22% vs 14%). Slightly fewer combined modality treatment patients had clinical N0 necks than the surgery alone group (20% vs 29%). Multivariate analysis was performed with the variables T, N stages, radiotherapy, margin status, primary tumor sites, microscopic and macroscopic extracapsular lymph node extension, number of positive lymph nodes, number of nodes with extracapsular lymph node extension. Locoregional control was maintained at 5 years in 59% of the combined modality treatment group and 31% of the surgery alone group (p.0001). Subgroup analysis likewise reveals significant differences favoring the combined modality treatment group for positive resection margins only (49% vs 41%; p = .04), extracapsular lymph node extension only (66% vs 31%; p = .03) and extracapsular lymph node extension+positive resection margins (68% vs 0%; p = .001). Adjusted survival also shows a significant benefit of combined modality treatment vs surgery alone for the entire group (72% vs 41%; p = .001). Multivariate analysis revealed that the use of radiotherapy is a strongly favorable variable for local control and adjusted survival. Macroscopic extracapsular lymph node penetration and positive resection margins are unfavorable independent variables for local control. T-stage is the only variable predicting local control in the combined modality group. Extracapsular extension remains an important negative prognostic variable for survival in both treatment groups. In conclusion, this study demonstrates a locoregional control and survival benefit for postoperative radiotherapy in patients with the high risk pathologic findings of extracapsular lymph node extension and positive resection margins.

Inhibition of the mitogen activated protein (MAP) kinase cascade potentiates cell killing by low dose ionizing radiation in A431 human squamous carcinoma cells

The molecular mechanism(s) by which tumor cells survive after exposure to ionizing radiation are not fully understood. Exposure of A431 cells to low doses of radiation (1 Gy) caused prolonged activations of the mitogen activated protein (MAP) kinase and stress activated protein (SAP) kinase pathways, and induced p21Cip-1/WAF1 via a MAP kinase dependent mechanism. In contrast, higher doses of radiation (6 Gy) caused a much weaker activation of the MAP kinase cascade, but a similar degree of SAP kinase cascade activation. In the presence of MAP kinase blockade by the specific MEK1 inhibitor (PD98059) the basal activity of the SAP kinase pathway was enhanced twofold, and the ability of a 1 Gy radiation exposure to activate the SAP kinase pathway was increased ∼sixfold 60 min after irradiation. In the presence of MAP kinase blockade by PD98059 the ability of a single 1 Gy exposure to cause double stranded DNA breaks (TUNEL assay) was enhanced at least threefold over the following 24–48 h. The increase in DNA damage within 48 h was also mirrored by a similar decrease in A431 cell growth as judged by MTT assays over the next 4–8 days following radiation exposure. This report demonstrates that the MAP kinase cascade is a key cytoprotective pathway in A431 human squamous carcinoma cells which is activated in response to clinically used doses of ionizng radiation. Inhibition of this pathway potentiates the ability of low dose radiation exposure to induce cell death in vitro.

The relative role of ErbB1–4 receptor tyrosine kinases in radiation signal transduction responses of human carcinoma cells

Activation of the epidermal growth receptor (ErbB1) occurs within minutes of a radiation exposure. Immediate downstream consequences of this activation are currently indistinguishable from those obtained with growth factors (GF), e.g. stimulation of the pro-proliferative mitogen-activated protein kinase (MAPK). To identify potential differences, the effects of GFs and radiation on other members of the ErbB family have been compared in mammary carcinoma cell lines differing in their ErbB expression profiles. Treatment of cells with EGF (ErbB1-specific) or heregulin (ErbB4-specific) resulted in a hierarchic transactivations of ErbB2 and ErbB3 dependent on GF binding specificity. In contrast, radiation indiscriminately activated all ErbB species with the activation profile reflecting that cells ErbB expression profile. Downstream consequences of these ErbB interactions were examined with MAPK after specifically inhibiting ErbB1 (or 4) with tyrphostin AG1478 or ErbB2 with tyrphostin AG825. MAPK activation by GFs or radiation was completely inhibited by AG1478 indicating total dependance on ErbB1 (or 4) depending on which ErbB is expressed. Inhibiting ErbB2 caused an enhanced MAPK response simulating an amplified ErbB1 (or 4) response. Thus ErbB2 is a modulator of ErbB1 (or 4) function leading to different MAPK response profiles to GF or radiation exposure.

Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells

In this study we have investigated the effects of low dose ionizing radiation (2 Gy) on p70 S6 kinase and Akt signaling with respect to Erb-B receptors in both the A431 squamous and the MDA-MB-231 mammary carcinoma cell lines. Ionizing radiation caused a 2–3-fold increase in p70 S6 kinase activity that was blocked pharmacologically using an EGFR inhibitor (AG1478) alone, or in combination with an Erb-B2 inhibitor (AG825). These results suggested that both EGFR and Erb-B2 receptors could initiate radiation-induced activation of p70 S6K. EGFR dependent Erb-B3 signaling also contributed to p70 S6 kinase activity through recruitment and activation of PI3K, which has been shown to regulate p70 S6 kinase activity. Furthermore, inhibition of the EGFR blocked IR stimulated increases in protein translation, a biologic consequence of p70 S6 kinase activation. We also report that ionizing radiation stimulated Akt activity that was partially independent of PI3K activity, but dependent on Erb-B2 function. Erb-B2 inhibition also correlated with enhanced apoptosis following IR exposure, suggesting an important role for Erb-B2 in cell survival. Together this work demonstrates that the Erb-B receptor tyrosine kinase network stimulates cytoprotective p70 S6 kinase and Akt activity in response to clinically relevant doses of ionizing radiation.

The tumor voluem and clonogen number relationship: Tumor control predictions based upon tumor volume estimates derived from computed tomography☆

PURPOSE While tumor volume is an important parameter predicting clinical outcome, its relationship to clonogen number remains uncertain. This uncertainty is related to many factors, among them treatment response heterogeneity, which obscures the influence of patients and treatment-related parameters. In this study, we analyze the effect of tumor volume on local and regional recurrence in a setting tightly controlled for dose, treatment time, and patient selection. The hypothesis that changes in clonogen number scale directly with changes in tumor volume is tested. METHODS AND MATERIALS Using digital reconstruction of diagnostic computed tomography (CT) scans, primary and total tumor volumes were estimated in 51 cases of advanced squamous cell carcinoma of the head and neck. All patients were managed with a concomitant boost accelerated superfractionated schedule to a median dose of 70.2 Gy. Clinical data were fitted to a mixture model to relate tumor volume parameters to control probability where volume and clonogen number were related by the relationship m = a.Vb, where m is initial clonogen number, a is a proportionality constant, V is tumor volume, and b is the volume exponent. RESULTS Tumor volume estimates for primary tumor ranged from 3-196 cm3 and for total tumor volume 5-196 cm3. Actuarial local-regional control is 63%. The estimated volume exponent b is 0.85 (95%, confidence interval (c.i.): 0.40-1.29) for primary tumor volume and 1.1 (95%, c.i.: 0.33-1.85) for total tumor volume. CONCLUSION This study quantifies the adverse influence of tumor volume on local-regional disease control in advanced head and neck cancer. The derived volume exponent approximates to one, the theoretical expectation if the growth fraction is roughly constant and clonogen number increases linearly with volume. Finally, these results suggest that radiobiological parameters are more reliably estimated from clinical data with narrowly defined strata.