Proliferation and micromilieu during fractionated irradiation of human FaDu squamous cell carcinoma in nude mice

PURPOSE: Previous functional radiobiological experiments demonstrated a significant acceleration of repopulation after 3 weeks and reoxygenation after 12 days of radiotherapy of FaDu tumours. Owing to the temporal coincidence between repopulation and reoxygenation, it was hypothesized that the improved oxygenation status during fractionated irradiation might be the preceding stimulus for increased proliferation. The study investigated whether these changes in repopulation and re-oxygenation are reflected by histological parameters of proliferation and the tumour micromilieu. MATERIALS AND METHODS: Human FaDu squamous cell carcinomas in nude mice were irradiated with three to 18 fractions of 3 Gy daily or every second day under normal blood flow and clamp hypoxia. At different time points, tumours were excised and stained for Ki67, BrdUrd, epidermal growth factor receptor (EGFR) and markers of the micromilieu (HOECHST 33342, pimonidazole, ER-MP12). RESULTS: On average, Ki67 and BrdUrd labelling indices decreased initially and increased again at later times during the course of fractionated radiotherapy. A similar kinetic pattern was found for the staining intensity of the EGFR. The vascular density in the viable tumour area remained constant during the whole course of irradiation, while the perfused fraction of vessels decreased within the first week of irradiation and returned to baseline values after 2 weeks. There was a corresponding increase in perfusion and a decrease in cellular hypoxia. CONCLUSIONS: The histological results were in surprisingly good agreement with the kinetics of clonogen repopulation and re-oxygenation determined previously using functional assays. The results support that the kinetics of repopulation of FaDu squamous cell carcinoma in response to fractionated irradiation are determined not only by intracellular processes, but also by a complex interaction of proliferation parameters with a changing microenvironment.     

Proliferation and micromilieu during fractionated irradiation of human FaDu squamous cell carcinoma in nude mice

Purpose: Previous functional radiobiological experiments demonstrated a significant acceleration of repopulation after 3 weeks and reoxygenation after 12 days of radiotherapy of FaDu tumours. Owing to the temporal coincidence between repopulation and reoxygenation, it was hypothesized that the improved oxygenation status during fractionated irradiation might be the preceding stimulus for increased proliferation. The study investigated whether these changes in repopulation and re‐oxygenation are reflected by histological parameters of proliferation and the tumour micromilieu.

Materials and methods: Human FaDu squamous cell carcinomas in nude mice were irradiated with three to 18 fractions of 3?Gy daily or every second day under normal blood flow and clamp hypoxia. At different time points, tumours were excised and stained for Ki67, BrdUrd, epidermal growth factor receptor (EGFR) and markers of the micromilieu (HOECHST 33342, pimonidazole, ER‐MP12).

Results: On average, Ki67 and BrdUrd labelling indices decreased initially and increased again at later times during the course of fractionated radiotherapy. A similar kinetic pattern was found for the staining intensity of the EGFR. The vascular density in the viable tumour area remained constant during the whole course of irradiation, while the perfused fraction of vessels decreased within the first week of irradiation and returned to baseline values after 2 weeks. There was a corresponding increase in perfusion and a decrease in cellular hypoxia.

Conclusions: The histological results were in surprisingly good agreement with the kinetics of clonogen repopulation and re‐oxygenation determined previously using functional assays. The results support that the kinetics of repopulation of FaDu squamous cell carcinoma in response to fractionated irradiation are determined not only by intracellular processes, but also by a complex interaction of proliferation parameters with a changing microenvironment.     

Cell kinetic changes in the follicular epithelium of pig skin after irradiation with single and fractionated doses of X rays

Changes in the cell kinetics of the follicular epithelium of the pig have been studied after irradiation with single and fractionated doses (30 fractions/39 days) of X rays and the results compared with previously published data for the epidermis. In the follicular epithelium there was an initial degenerative phase, during which the rate of cell depletion was independent of the radiation dose and the mode of administration. Evidence for repopulation was seen between the 14th and 18th days after single doses (15 or 20 Gy) and by the 28th day after the start of irradiation with fractionated doses (52.3-80.0 Gy). However, the degree of cell depletion and the subsequent rate of repopulation were independent of dose. The regenerative phase was characterized by an increased cell proliferation as indicated by an elevation of the labelling index. Islands of cells (colonies), with an appearance similar to cells in the normal follicular epithelium, were seen 18 days after a single dose of 20 Gy and 42 days after the start of fractionated irradiation. When compared with the epidermis, the follicular epithelium exhibited considerably less evidence of damage after both single and fractionated doses of X rays. There was a lower incidence of degenerate cells and reduced levels of cell depletion in the follicular epithelium, suggesting that cells from this region play an important role in the repopulation of the epidermis after high-dose irradiation.     

Differentiation status of human squamous cell carcinoma xenografts does not appear to correlate with the repopulation capacity of clonogenic tumour cells during fractionated irradiation

PURPOSE: To investigate the magnitude and kinetics of repopulation in a moderately well differentiated UT-SCC-14 human squamous cell carcinoma [hSCC] in nude mice. This question is of interest because clinical data indicate a higher repopulation capacity in those SCC that have preserved characteristics of differentiation, which appears to be in contrast to results on FaDu and GL hSCC previously reported from this laboratory. METHODS AND MATERIALS: UT-SCC-14 tumours were transplanted subcutaneously into the right hind leg of NMRI nu/nu mice. Fractionated radiation treatments were delivered, either under clamped hypoxia at 5.4 Gy/fraction or under ambient conditions (consistent with an OER of 2.7). Tumours were irradiated every day, every 2nd day, or every 3rd day with 6, 12 or 18 fractions. 1, 2 or 3 days after the last fraction, graded top-up-doses under clamped conditions were given for the purpose of estimating the 50% tumour control dose (TCD50). A total of 22 TCD50 assays were performed and analysed using maximum likelihood techniques. RESULTS: The data demonstrate a slow but significant repopulation of clonogenic cells during fractionated irradiation of UT-SCC-14 hSCC. The results under hypoxic conditions are consistent with a constant repopulation rate, with a clonogenic doubling time (Tclon) of 15.6 days (95% CI: 9.7, 21.4). This contrasts with ambient conditions where Tclon was 68.5 days (95% CI: 124, 161). Both Tclon values are longer than the 6-day volume doubling time of untreated tumours. CONCLUSIONS: Less pronounced repopulation for irradiation under ambient compared to clamped hypoxic conditions might be explained by preferential survival of hypoxic and therefore non-proliferating clonogenic cells. Taken together with previous studies on poorly differentiated FaDu and moderately well differentiated GL hSCC, the results are consistent with considerable variability in the magnitude and kinetics of repopulation in different experimental squamous cell carcinomas, and with a relationship between reoxygenation and repopulation during fractionated irradiation. The differentiation status of hSCC growing in nude mice does not to appear to correlate with the proliferative capacity of clonogenic tumour cells during treatment. The results do not support the hypothesis gained from clinical data of higher repopulation in well-differentiated tumours.     

Differentiation status of human squamous cell carcinoma xenografts does not appear to correlate with the repopulation capacity of clonogenic tumour cells during fractionated irradiation

Purpose: To investigate the magnitude and kinetics of repopulation in a moderately well differentiated UT‐SCC‐14 human squamous cell carcinoma [hSCC] in nude mice. This question is of interest because clinical data indicate a higher repopulation capacity in those SCC that have preserved characteristics of differentiation, which appears to be in contrast to results on FaDu and GL hSCC previously reported from this laboratory.

Methods and Materials: UT‐SCC‐14 tumours were transplanted subcutaneously into the right hind leg of NMRI nu/nu mice. Fractionated radiation treatments were delivered, either under clamped hypoxia at 5.4?Gy/fraction or under ambient conditions (consistent with an OER of 2.7). Tumours were irradiated every day, every 2nd day, or every 3rd day with 6, 12 or 18 fractions. 1, 2 or 3 days after the last fraction, graded top‐up‐doses under clamped conditions were given for the purpose of estimating the 50% tumour control dose (TCD₅₀). A total of 22 TCD₅₀ assays were performed and analysed using maximum likelihood techniques.

Results: The data demonstrate a slow but significant repopulation of clonogenic cells during fractionated irradiation of UT‐SCC‐14 hSCC. The results under hypoxic conditions are consistent with a constant repopulation rate, with a clonogenic doubling time (T_clon) of 15.6 days (95% CI: 9.7, 21.4). This contrasts with ambient conditions where T_clon was 68.5 days (95% CI: 124, 161). Both T_clon values are longer than the 6‐day volume doubling time of untreated tumours.

Conclusions: Less pronounced repopulation for irradiation under ambient compared to clamped hypoxic conditions might be explained by preferential survival of hypoxic and therefore non‐proliferating clonogenic cells. Taken together with previous studies on poorly differentiated FaDu and moderately well differentiated GL hSCC, the results are consistent with considerable variability in the magnitude and kinetics of repopulation in different experimental squamous cell carcinomas, and with a relationship between reoxygenation and repopulation during fractionated irradiation. The differentiation status of hSCC growing in nude mice does not to appear to correlate with the proliferative capacity of clonogenic tumour cells during treatment. The results do not support the hypothesis gained from clinical data of higher repopulation in well‐differentiated tumours.     

VEGF和Ang-1在皮肤创伤和放射性烧伤愈合过程中的表达

目的研究血管内皮生长因子（VEGF）和促血管生成素-1（Ang-1）在皮肤单纯性创伤和放射性烧伤中表达的动态变化,了解其在血管再生中的作用,为深入探讨放射性烧伤的愈合机制及其治疗提供有意义的实验资料。方法在大鼠背侧切开皮肤全层制备大鼠创伤模型（单创组）,应用电子线照射臀部皮肤,建立放射性烧伤模型（放伤组）。于伤后1-60d不同时间活体伤口取材,HE染色组织病理学观察,应用Western blot法检测VEGF和Ang-1蛋白的表达。结果在单纯性皮肤创伤愈合过程中,VEGF和Ang-1蛋白表达变化均在伤后第1天开始上升,伤后第5-7天达高峰,然后逐渐下降到接近正常水平。在放射性烧伤愈合过程中,VEGF是先降低后升高然后再降低,而Ang-1的变化与此相反。与单创组比较,放烧组VEGF蛋白表达在第1、7和14天明显降低,差异有统计学意义（P〈0.01）;在第1、7、14、21、28和35天Ang-1蛋白表达明显降低,差异有统计学意义（P〈0.01或P〈0.05）,此后渐升高。结论放射性烧伤愈合早期阶段VEGF和Ang-1的蛋白表达明显降低,是放射性烧伤愈合延迟的重要原因之一。     

放疗对人宫颈癌组织的增殖细胞核抗原(PCNA)和c-fos表达的影响

目的：探讨照射前、后人宫颈组织产殖细胞核抗原（PCNA）和c-fos表达的变化。方法：在多聚甲醛固定的活检癌组织冰切片上,进行PCNA和Fos的免疫组织化学染色。结果：放疗前的宫颈癌组织内,多数癌细胞呈PCNA免疫染色阳性;放疗后,多数癌细胞无PCNA染色,但间质细胞的PCNA免疫染色明显升高,大量间质细胞则呈中等或强阳性染色。放射治疗前的宫颈癌组织内,多数癌细胞呈Fos免疫染色阳性,放疗后的宫颈癌组织Fos免疫染色显著降低,多数癌细胞无Fos染色,但放疗前、后间质细胞的Fos免疫反应无明显变化。结论：照射抑制宫颈癌细胞的PCNA和c-fos表达的同时,诱导间质细胞的PCNA表达,提示PCNA和c-fos的表达状态可作为放射治疗中评价宫颈癌细胞增殖和间质细胞修复的分子指标之一。     

Effects of keratinocyte growth factor in the squamous epithelium of the upper aerodigestive tract of normal and irradiated mice.

PURPOSE: To investigate the effects of keratinocyte growth factor (KGF) on the structure of the stratified squamous epithelium of the tongue, buccal mucosa and oesophagus of normal and irradiated mice. MATERIALS AND METHODS: Female BDF1 mice were exposed to total body irradiation from a caesium source. The irradiated mice and normal, unirradiated mice were injected with 5 mg/kg per day KGF or vehicle. Thickness and proliferation in the epithelium were measured. RESULTS: KGF caused epithelial thickening of the non-keratinized layers in oral epithelium in normal mice. It increased the number of nucleated layers and influenced differentiation of post-mitotic cells in the upper layers by increasing the size and number of keratohyalin granules, and the number of desmosomes. Single and fractionated doses of radiation caused inhibition of proliferation as detected by markedly reduced BrdU incorporation following exposure, followed by epithelial atrophy. KGF treatment of mice reversed the inhibition of proliferation and atrophy that occurred in control irradiated mice. CONCLUSION: These data show that KGF reverses epithelial atrophy in mouse oral cavity caused by irradiation and suggest that KGF may be useful for the treatment of mucositis of the upper aerodigestive tract of patients treated with aggressive regimens of radiation therapy.     

CD44分子在正常皮肤和银屑病表皮的表达

采用免疫组化染色研究正常人皮肤的CD44表达情况，发现CD44有选择性地存在于皮肤的不同部位，表皮中基底细胞及棘细胞表面有CD44，而颗粒层及角质层均无CD44。真皮组织无CD44，而毛囊外根鞘，皮脂腺毛囊口及外分泌汗腺均有CD44表达，测定了银屑病患者表皮的CD44，结果是损害处表皮的棘细胞及基底细胞表面CD44比无损害的治愈后的表皮要显著得多，用逆转录聚合酶链反应（RT-PCR）分析CD44的类型，结果为CD44的标准型。     

Early and long-term effects of radiation on intercellular adhesion molecule 1 (ICAM-1) expression in mouse urinary bladder endothelium

The aim was to assess the effect of irradiation on intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells of vessels in mouse urinary bladder and to compare endothelial ICAM-1 expression with changes in bladder function (storage capacity) during the early and late radiation response phases. Female C3H/Neu mice were irradiated with doses of either 20 or 0 Gy. For assessment of ICAM-1 expression, which was measured by the intensity of the immunohistochemical staining signal in bladder endothelium, an arbitrary semiquantitative score (0 - 3) was applied. Bladder storage function was assessed by transurethral cystotonometry. A positive functional radiation response, defined as a reduction in bladder capacity by > 50%, between days 0 and 15 or 16 and 30 was found in 40 and 64% of the animals, respectively. A late functional response was observed in 71% of the animals sacrificed after day 180. Minor constitutive expression of ICAM-1 was observed in bladder endothelial cells. After irradiation, an increase in staining signal by day 2, with a maximum on day 4, and on days 16 - 28 was found, which preceded the functional radiation effects. A permanent increase in ICAM-1 staining signal was observed in the late phase on top of an age-related rise. ICAM-1 expression was significantly higher in animals with a positive late response on day 90, i.e. during the initial late phase. Irradiation induces significant early and chronic variations in ICAM-1 expression in bladder endothelium, which preceded the functional response. This suggests that endothelial ICAM-1 is involved in the pathogenesis of both the early and late phases of radiation-induced urinary bladder effects.     

Early and long-term effects of radiation on intercellular adhesion molecule 1 (ICAM-1) expression in mouse urinary bladder endothelium

The aim was to assess the effect of irradiation on intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells of vessels in mouse urinary bladder and to compare endothelial ICAM-1 expression with changes in bladder function (storage capacity) during the early and late radiation response phases. Female C3H/Neu mice were irradiated with doses of either 20 or 0?Gy. For assessment of ICAM-1 expression, which was measured by the intensity of the immunohistochemical staining signal in bladder endothelium, an arbitrary semiquantitative score (0?–?3) was applied. Bladder storage function was assessed by transurethral cystotonometry. A positive functional radiation response, defined as a reduction in bladder capacity by >?50%, between days 0 and 15 or 16 and 30 was found in 40 and 64% of the animals, respectively. A late functional response was observed in 71% of the animals sacrificed after day 180. Minor constitutive expression of ICAM-1 was observed in bladder endothelial cells. After irradiation, an increase in staining signal by day 2, with a maximum on day 4, and on days 16?–?28 was found, which preceded the functional radiation effects. A permanent increase in ICAM-1 staining signal was observed in the late phase on top of an age-related rise. ICAM-1 expression was significantly higher in animals with a positive late response on day 90, i.e. during the initial late phase. Irradiation induces significant early and chronic variations in ICAM-1 expression in bladder endothelium, which preceded the functional response. This suggests that endothelial ICAM-1 is involved in the pathogenesis of both the early and late phases of radiation-induced urinary bladder effects.     

Radiation-induced variations in urothelial expression of intercellular adhesion molecule 1 (ICAM-1): association with changes in urinary bladder function

PURPOSE: To assess the effect of single-dose irradiation on intercellular adhesion molecule 1 (ICAM-1) expression in the urothelium of mouse urinary bladder and to correlate ICAM-1 variations with fluctuations in storage capacity during the early and late radiation response. MATERIALS AND METHODS: Groups of female C3H mice were subjected to irradiation with either 20 or 0 Gy. The intensity of immunohistochemical ICAM-1 staining in the urothelium was assessed in a semiquantitative way applying an arbitrary score (0-5). Changes in bladder storage function were assessed by transurethral cystometry. RESULTS: For the early radiation response phase, a reduction in bladder capacity by >50%, i.e. a positive functional radiation response, was seen in 40% of the irradiated animals between days 0 and 15, and in 64% of animals during days 16-30. During the late response phase, 71% of the animals sacrificed after day 180 developed a positive functional response. Urothelial cells were found to express ICAM-1 constitutively. Irradiation resulted in an early rise in staining signal by day 2, with a maximum on day 4 and a return to control values on day 13. A permanent increase in ICAM-1 staining signal was observed in the late phase, from day 90 to 360 after irradiation. The expression of ICAM-1 in animals with a positive late response was 4.2+/-1.2 (mean+/-standard deviation), compared with 2.6+/-1.0 in non-responders (p=0.0009). CONCLUSION: Irradiation induces significant acute and chronic changes in urothelial ICAM-1 expression indicating that the urothelium contributes to the pathogenesis of both acute and late radiation effects in the urinary bladder.     

Modulation of repopulation processes in oral mucosa: experimental results

PURPOSE: Repopulation processes, i.e. the tissue regeneration responses to radiotherapy with increasing overall treatment time, are the predominant factors defining the radiation tolerance of turnover tissues, such as squamous epithelia of the skin or gastrointestinal tract. The purpose was to assess experimental approaches for the modulation, i.e. stimulation of repopulation, in normal oral mucosa. MATERIALS AND METHODS: Numerous studies have been performed to identify and quantify the efficacy of repopulation processes in oral mucosa in experimental animal models, mainly mouse lip and tongue mucosa, and in some clinical studies. However, only a few investigations focused on the stimulation of these processes, aiming at a reduction in oral mucosal side-effects of radiotherapy. The present review summarizes the biological mechanisms underlying effective repopulation, and delineates experimental approaches for effective stimulation of these processes, eventually resulting in an increase in oral mucosal radiation tolerance. RESULTS: Repopulation in oral mucosa is a complex process dominated by a substantial reorganization of the proliferative structure, including a loss of the stem cell division asymmetry and acceleration of stem cell proliferation, as well as abortive divisions of 'sterilized' cells. Repopulation in mouse oral mucosa is more effective if the initial dose intensity (weekly dose) during the first treatment week(s) is increased. Stem cell production occurs mainly during the treatment weeks, while during treatment breaks, including weekends, differentiating (transit) cells are preferentially produced. Stimulation of superficial cell loss, e.g. by topical administration of mild astringent agents, stimulates mucosal proliferation. This translates into increased radiation tolerance to fractionated irradiation in experimental systems, like mouse tongue mucosa. In clinical studies, a reduction of oral mucosal reactions using the same approach was found during an accelerated radiotherapy regimen, but not during conventionally fractionated protocols. Keratinocyte growth factor has been demonstrated to reduce oral mucosal reactions significantly to single dose and fractionated irradiation. This effect is presumably based on an interaction with repopulation processes. CONCLUSIONS: Repopulation in oral mucosa is a highly complex process, which includes a substantial reorganization of the proliferative structure. In experimental models, its efficacy can be modulated by changes in the fractionation protocol, but more effectively by intervention in the regulation processes, e.g. by stimulation of proliferation through enhancement of cell loss. An alternative promising approach is the administration of growth factors, like keratinocyte growth factor, for effective modulation of oral mucosal repopulation. However, selectivity for the normal tissue, as well as biological mechanisms, must be studied in detail in relevant animal models.     

Some radiobiological aspects of total body irradiation (TBI)

Data from the literature on total body irradiation are reported. The biological concept of fractionation of TBI is reviewed. The therapeutic gain of fractionated single dose as compared single dose to irradiation is explained. However, other data from Song et al. show that repair capacity of hemopoietic or leukemic cells may be underestimated. More data with fractionation experiments are needed. The significance of repopulation between fractionation doses is unknown. The interaction of TBI and preceding chemotherapy should be studied in more detail.     

Expression of oestrogen receptor and transforming growth factor-alpha in MCF-7 cells after exposure to fractionated irradiation.

The expression of critical growth controlling genes was studied in MCF-7 cells after exposure to cumulative radiation doses of 20 and 60 Gy yielding cell lines called MCF-IR-1 and MCF-IR-3, respectively. The irradiated cell lines exhibited increased plating efficiencies but no differences in growth rates. MCF-IR-1/-IR-3 cells showed a reduced oestrogen responsiveness as indicated by their diminished response to tamoxifen-induced growth arrest and 17 beta-oestradiol (E2)-induced growth stimulation. The reduced expression of oestrogen receptor (ER) was determined by quantitative immune peroxidase staining of single cells and by total cellular E2 binding. There was also a radiation dose-dependent increase in the radiosensitivity of MCF-IR-3 cells as determined by the radiobiological parameters alpha, beta, and D (mean inactivation dose). Using RNA protection assays the irradiated cell lines produced steady-state ER mRNA at reduced levels while the levels of TGF-alpha were unchanged in MCF-IR-1 cells but increased 2.8-fold in MCF-IR-3 cells. A similar pattern was seen for TGF-alpha protein. While the current analyses cannot differentiate between radiation-induced altered gene expression or cell selection the results demonstrate that reduced ER expression and increased TGF-alpha expression are associated with the survival of MCF-7 cells after fractionated irradiation in vitro. In contrast, the MCF-IR cells were found to be more radiosensitive in acute survival experiments.     

Changes in the cell kinetics of pig epidermis after single doses of X rays

Changes in the cell kinetics of pig epidermis have been investigated after irradiation with single doses of 15 Gy and 20 Gy of X rays. The epidermis exhibited an initial degenerative phase when the rate of cell depletion was independent of radiation dose. Changes consistent with repopulation were evident between the 14th and 18th day after irradiation. The severity of cell depletion and the rate of recovery of the epidermis were dose dependent. The regenerative phase was characterized by an increased cell proliferation; values for the labelling index (LI) were greater than those in the non-irradiated epidermis, from 14 days after 15 Gy and from 18 days after 20 Gy. The LI was still elevated at the end of the observation period, i.e. Day 56. No change in the time for DNA synthesis was found. Eighteen days after 15 Gy and 22 days after 20 Gy, islands of cells (colonies), with an appearance similar to the cells in the normal epidermis, were seen. The minimum turnover time (TT) for the proliferating cells of the basal layer of the epidermis in radiation-damaged skin was 61-68 h as compared with 125 h in unirradiated skin. For the basal cells in the colonies, TT was 16-22 h.     

Analysis of cell kinetics after irradiation by flow cytometry (II)--Evaluation of clonogenic potential of G2 block cells after irradiation by cell sorting

When the tumor cells are irradiated, the percentage of the cells in the G2 + M phase and the duration of the cell cycle arrested increase with the irradiation dose. This post irradiation growth inhibition and changes in the cell kinetics observed within 48 hours of irradiation are closely correlated. Cells in the G2 + M phase are reported to be sensitive to irradiation, but little is known about their clonogenic potential. These cells must be separated for evaluation of their clonogenic potential. In this study, the clonogenic potential of irradiated G2 phase arrested cells was examined by the flow cytometric cell sorting technique. Fluorescent Hoechst staining, which allows determination of DNA content in living cells, was used. The results of DNA assay using Hoechst 33342 agreed well with those using PI staining. The concentration of Hoechst 33342 required for the DNA assay and cell sorting is 5 micrograms at which vital staining is possible with 80% cell viability. Although the clonogenic potential of non-irradiated cells was not different between those in G1 and G2 + M phases, that of the cells arrested in the G2 + M phase after irradiation was lower than that of the cells in the G1 phase. The reduction in the clonogenic potential was more notable in the cells subjected to high dose irradiation. A greater number of cells were arrested for a longer period than the cells irradiated at a lower dose. The clonogenic potential of the cells correlated well with the duration of G2 block.