Gene expression changes during repopulation in a head and neck cancer xenograft

Background/purposeTo investigate temporal changes in global gene expression and pathways involved in the response to irradiation during phases of growth inhibition, recovery and repopulation in a human head and neck squamous cell cancer (HNSCC) xenograft.Methods and materialsLow passage head and neck squamous cancer cells (UT-14-SCC) were injected into the flanks of female nu/nu mice to generate xenografts. After tumors reached a size of 500 mm³, they were treated with either sham RT or 15 Gy in one fraction. At different time points, days 0, 3, and 10 for controls and days 4, 7, 12, and 21 after irradiation, the tumors were harvested for global gene expression analysis and pathway analysis.ResultsThe tumors showed growth inhibition through days 4–7 and began the transition to regrowth around the day 12 time point. When comparing the pooled controls to each day of treatment, there were 22, 119, 125, and 25 differentially expressed genes on days 4, 7, 12, and 21 respectively using a p ⩽ 0.01 and a 2-fold cut-off. Gene Ontology (GO), gene set enrichment analysis (GSEA) and sub-network enrichment analysis (SNEA) identified different biological processes, cell process pathways and expression targets to be active on each time point after irradiation. An important observation was that the molecular events on day 12 which represented the transition from growth inhibition to regrowth identified interferon and cytokine related genes and signaling pathways as the most prominent.ConclusionThe findings in this study compliment research which has identified components of interferon-related signaling pathways to be involved in radioresistance. Further work will be required to understand the significance of these genes in both radioresistance and treatment response leading to new therapeutic strategies and prognostic tools.     

Histological observations on the regrowth of malignant gliomas after radiotherapy and chemotherapy

Summary The reproliferation of glioblastomas after radiation and chemotherapy has been studied in 25 tumors by means of whole mount histological preparations. The tumors have been subdivided into four groups according to the radiation dose and the distance from the end of radiation. After 4,000 rad vessel proliferations and mitoses stop, while vessel degenerations and astrocytes with deformed nuclei appear. Six months after 6,000 rad have been delivered, repopulation phenomena are clearly evident and are mainly represented by mitoses both in parenchyma and in the vessel walls, circumscribed necroses with pseudopalisadings, proliferations of formerly degenerated vessels, etc.The brain adjacent to tumor (BAT) has a great importance since it is one of the reproliferating sites, even though it may be unrecognizable for the occurrence of radiation damages.     

The influence of overall treatment time on renal injury after multifraction irradiation

The influence of overall treatment time on the radiation response of the mouse kidney was studied in an experiment in which 16 fractions were administered either evenly distributed over 20, 40 or 80 days, or as a split course (8 F/3 days; 74 days rest; 8 F/3 days). Urine output and an isotope assay of glomerular filtration were used to test the mice sequentially. The data were used both to obtain dose-response curves and also to determine the latent period before a chosen level of injury was expressed functionally. Prolonging the overall time from 20 to 80 days increased the isoeffect dose by 2-5 Gy (4-9%) for the isotope assay, and by 4-9 Gy (7-18%) for the urine output assay. This additional recovery as the interval between fractions was prolonged from 1 to 5 days is consistent with slow repair and can be expressed as a small "T" exponent of 0.02-0.12. (One analysis gave a result consistent with negative repair, but the errors on this result were unusually wide.) When the radiation was given as a split course, at the rate of 2 fractions per day, with a large gap of 10.5 weeks between courses, there was no additional sparing compared with 16 fractions over 20 days. This indicates that any sparing that might have resulted from slow repair or stimulated repopulation in the gap has been counterbalanced by having less time for repair of sublethal injury when intervals of 6-12 h are used instead of 24-48 h. Clearly no great increase in the tolerance dose for mouse kidney resulted from the split course.     

Adriamycin sensitivity following "ultra" low dose rate irradiation of Lewis lung carcinoma in vivo

As an extension of recent study on the response of the Lewis lung tumor to low dose rate continuous irradiation (CI) at 15 cGy/hr, we have gone on to investigate the effects of such irradiation on the sensitivity of tumor cells to treatment with Adriamycin (Adr). Cells from untreated tumors gave an exponential dose response curve to Adr in vitro, the D10 of which increased (sensitivity decreased) with the size of tumor (0.05 g to 0.6 g) from which the cells were obtained. After previous in vivo CI to a total dose of 28 Gy (irradiation time--186 hr), this size dependence was abolished and the cells showed an exponential response to Adr in vitro (D10 = 0.4 microgram/ml). The enhancement was also observed after equivalent doses of fractionated irradiation, but not after acute irradiation. It was difficult to characterize the proliferative status of the cells surviving irradiation, but repopulation studies showed that only after CI was there any delay before repopulation commenced. LL was relatively insensitive to Adr in vivo, however, we did observe an increased effect after previous CI.     

External-beam radiotherapy as preparative regimen for hepatocyte transplantation after partial hepatectomy

PURPOSE: The transplantation of donor hepatocytes is considered a promising option to correct chronic liver failure through repopulation of the diseased organ. This study describes a novel selective external-beam irradiation technique as a preparative regimen for hepatocyte transplantation. METHODS AND MATERIALS: Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with external-beam single-dose irradiation (25 Gy) delivered to two thirds of the liver. Four days later, a one-third partial hepatectomy (PH) was performed to resect the untreated liver section, and 15 million wild-type (DPPIV+) hepatocytes were transplanted via the spleen into the recipient livers. The degree of donor-cell integration and growth was studied 8 h, 3 days, and 5 and 12 weeks after transplantation. RESULTS: Transplanted hepatocytes integrated rapidly into the irradiated liver and proliferated as clusters, finally repopulating the host liver to approximately 20% hepatocyte mass. After 12 weeks, donor cells and their numerous descendents were fully integrated and expressed functional markers to the same extent as host hepatocytes. CONCLUSIONS: We demonstrate that external-beam liver irradiation is sufficient to achieve partial repopulation of the host liver after hepatocyte transplantation, under the additional stimulus of one-third PH. The method described has potentially good prospects for its application in a clinically viable form of treatment.     

细胞周期基因芯片筛选不同增殖状态下CNE-2细胞的差异表达基因

目的：了解不同增殖状态鼻咽癌细胞（CNE-2）细胞周期基因表达差异，探讨鼻咽癌放射治疗中加速再增殖的机理。方法：对不同增殖状态的CNE-2细胞，采用基因芯片技术、实时荧光定量PCR检测其细胞周期相关基因表达差异。结果：细胞增殖活性最高时相（2Gy连续照射第3天）与最低时相（2Gy连续照射第5天）表达有差异的基因有3条，为检测基因总数的3％（3／100），其中细胞增殖活性增高时上调基因为CCNE1、CUL-5，下调基因为CDKN1A。应用相对定量荧光实时PCR技术对部分差异表达在2倍以上的基因（CCNE1、CDKN1A））进行了mRNA水平的验证，结果显示实时PCR实验结果与芯片结果具有良好的一致性。结论：某些细胞周期基因如CCNE1、CUL-5、CDKN1A等可能参与了鼻咽癌放射治疗过程中发生的加速再增殖过程，有必要对这些基因功能作进一步研究，以探明照射过程中肿瘤细胞加速再增殖发生的分子机制。     

The influence of the number of fractions and of overall treatment time on local control and late complication rate in squamous cell carcinoma of the larynx

Three hundred and ten patients with $\text{T}{}_3\text{T}{}_4$ N₀, M₀ squamous cell carcinoma were irradiated with 200 kV X rays with total doses ranging from 4,900 to 6,200 rad, given in 21 to 35 fractions in 32–63 days. After a minimum follow-up period of 3 years, the local control rate was 50% ; 21 severe late complications were observed among the patients. The dependence of local control rate and of late complication rate on the dose per fraction and on overall treatment time was analyzed by various statistical methods. Whereas the late complication rate depended significantly on dose per fraction, local tumor control depended strongly on overall treatment time.     

Continuous irradiation of the lewis lung carcinoma in vivo at clinically-used “ultra” low-dose-rates

The in vivo response of the transplantable Lewis lung tumor to continuous irradiation (CI) at the clinical “ultra” low-dose-rates of 10–30 cGy/h was investigated. Tumor-bearing mice were housed in a dedicated ¹³⁷Cs unit for up to three weeks while receiving Cl. The horizontal ¹³⁷Cs beam allowed secondary shielding of the lower body, but full irradiation of the dorsally-placed tumor and part of the upper body. Radiation survival curves were measured using an excision assay and tumor-cell colony formation in soft agar. The relative cell survival per tumor decreased exponentially with time and the irradiation periods required to reach a surviving fraction of 10^?3 at the three dose-rates (10, 15 and 30 cGy/h) were 15.4, 10.2 and 4.3 days. As a function of dose, the survival curves were exponential and indistinguishable betwen the three dose-rates, the aveage D₀ value being 5.04 Gy. Neither the hypoxic fraction of the tumor nor the intrinsic cellular radiosensitivity were altered in the tumor cells surviving Cl. The effects of the three dose-rates on the growth of Lewis lung tumors differed considerably, with clear volume regression at the highest dose rate (30 cGy/h) and exponential decreases in clonogenic cells per tumor with time at 15 and 30 cGy/h. Since the overall effect of CI is determined by the competition between cell killing and cell repopulation, the response of slowly-growing human tumors should be proportionately greater.     

Cumulative interfraction interval analysis of influence of time and interruptions on radiotherapy results in oral cancers

To present a new approach to analyze the influence of time and interruptions in fractionated radiotherapy, in terms of cumulative interfraction intervals (CIFIs), that is, the interval between nonsuccessive fractions of radiotherapy.

The subjects were 142 patients with epidermoid cancer of the buccal mucosa-gingiva-palate region treated by 60 Gy in 25 fractions during 5 weeks. The influence of CIFI between the first and sixth, eleventh, sixteenth, and twenty-first fractions (CIFI_1–6, CIFI_1–11, CIFI_1–16, and CIFI_1–21, respectively), as well as overall treatment time on local control, was analyzed by Kaplan-Meier calculation of the primary relapse-free survival and Mann-Whitney U testing of the difference in various CIFIs between the tumors that recurred (recurred group) and those that did not (controlled group).

Only CIFI_1–11 influenced local control significantly, with the 5-year primary relapse-free survival rate being 67.9%, 48.4%, and 32.6% in those with CIFI_1–11 of <14, 14, and >14 days (p = 0.0181). The median CIFI_1–11 in the controlled group was significantly lower than that in the recurred group (14 days vs. 15 days; p = 0.0037).

Interruptions during the first 11 fractions, including planned weekend gaps, decrease the effect of radiotherapy, possibly because of successful tumor cell repopulation during the protracted interfraction intervals. The analysis of time in terms of CIFI rather than overall treatment time appears to be a promising area for research.     

环磷酰胺对睾丸组织中SCF表达的影响及意义

目的：通过环磷酰胺对大鼠睾丸组织中干细胞因子的表达及增殖指数影响的研究,以探讨环磷酰胺所致生精功能损害的原因.方法：选取9天龄雄性Wistar大鼠,腹腔注射大、中、小剂量的环磷酰胺后24h、4w、8w分别采集睾丸标本,应用RT-PCR技术检测睾丸组织中SCF的表达,同时光镜下计数4w时睾丸组织的增殖指数.结果：（1）对照组随年龄的增加,睾丸组织中分泌型干细胞因子表达无显著差异（P＞0.05）,膜型干细胞因子均有显著增加（P＜0.05）.（2）同一时期各实验组和对照组比较,分泌型干细胞因子表达无显著差异（P＞0.05）,但膜型干细胞因子表达均有显著降低（P＜0.05）;同一时期各实验组间比较,随环磷酰胺剂量增加,膜型干细胞因子表达有显著降低（P＜0.05）.（3）同一剂量三个时期比较,各实验组分泌型干细胞因子无显著差异（P＞0.05）;膜型干细胞因子比较,大剂量组中无显著差异（P＞0.05）,中、小剂量组中24h较4w、8w均有显著降低（P＜0.05）,4w与8w比较无显著差异（P＞0.05）.（4）增殖指数检测,4w时各实验组与对照组比较,均有显著降低（P＜0.01）,并与剂量负相关.结论：本研究结果提示由于环磷酰胺的毒性作用,导致了睾丸组织中膜型干细胞因子表达下调,进而影响增殖指数降低,这可能即是临床上环磷酰胺使用后产生不同程度生精功能障碍的原因之一.