Short-Term Effects of Early-Acting and Multilineage HematoPoietic Growth Factors on the Repair and Proliferation of Irradiated Pure Cord Blood (CB) CD34 Hematopoietic Progenitor Cells

Purpose: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation.

Methods and Materials: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34⁺ cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34⁺ cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose–response curves were fitted to the data.

Results: The radiobiological parameters (D₀ and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34⁺ cells compared to optimal two-factor combinations. The D₀ values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56–1.15, 0.41–2.24, and 0.56–1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n = 1 for all tested GF(s). DNA repair capacity of CD34⁺ cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34⁺ cells was similar.

Conclusion: Our data indicate that increased survival of irradiated CB CD34⁺ cells after short-term GF treatment is mediated through proliferative GF effects on the surviving fraction but not through improved DNA repair capacity.     

Chromosomal aberrations induced by chemotherapy and radiotherapy in lymphocytes from patients with breast carcinoma

: Stable chromosomal aberrations (SCAs) have been found in circulating lymphocytes from patients treated for breast carcinoma. Therefore, we tried to define their incidence in such patients, to determine an in vitro dose-effect relationship, and to correlate these data with clinical parameters.

: This prospective study included 25 patients who, after surgery, underwent either radiotherapy (RT) alone (n = 15) or RT combined with chemotherapy (n = 10). SCAs were scored using the fluorescent in situ hybridization technique before RT and 4 and 12 months after RT. Dose-effect curves were established by in vitro irradiation of blood samples with 2 and 4 Gy, before and after treatment.

: In all patients, the rate of SCAs increased significantly after external irradiation. No significant decrease in SCAs was observed during the first year after RT. RT and chemotherapy had no effect on the lymphocyte in vitro dose-effect relationship. No relationship was found in the distribution of patients between the yield of SCAs scored after external irradiation and after in vitro irradiation. SCAs after RT or in vitro irradiation did not correlate with family history of breast carcinoma or acute toxicity of treatment. More significantly, the yield of SCA after external irradiation was strongly related to the irradiation of the internal mammary chain and the supraclavicular lymph node area, suggesting that the volume of irradiated blood vessels was an essential parameter in determining the rate of SCAs.

: A high and stable yield of SCAs persisted at least 1 year after external irradiation. The nature of the volume irradiated containing large blood vessels was the major determinant of the observed biologic dose.     

Abortive Mitoses and Nuclear DNA Fragmentation in CD30+ Large Cells of Hodgkin's Disease

This study was undertaken to better comprehend the reasons for the scarcity of Hodgkin and Reed-Sternberg (H-RS) cells in Hodgkin's disease (HD) despite their expression of “proliferation-associated antigens”. To this end, we assessed the relative frequency of mitotic phases and nuclear damage (detected by in situ end-labeling of DNA strand breaks) in CD30⁺ large cells of nodular sclerosis and mixed cellularity HD. Our results show that a) most CD30⁺ cells in HD exhibit abortive mitoses, with a highly significant arrest at the metaphase-ana/telophase transition, and b) many of these elements, i.e. mainly H-RS cells, show fragmentation of nuclear DNA, suggesting imminent or actual death. Percentages of CD30⁺ cells that entered mitosis and those with DNA strand breaks were of a similar order of magnitude and correlated significantly in a linear fashion. These findings are consistent with the concept that cell deletion is the major cause of the paucity of H-RS cells in HD.     

Radiation-induced apoptosis in different pH environments in vitro

: The effect of environmental pH on the radiation-induced apoptosis in tumor cells in vitro was investigated.

: Mammary adenocarcinoma cells of A/J mice (SCK cells) were irradiated with γ-rays using a ¹³⁷Cs irradiator and incubated in media of different pHs. After incubation at 37°C for 24–120 h the extent of apoptosis was determined using agarose gel electrophoresis, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, flow cytometry, and release of ³H from ³H-thymidine labeled cells. The clonogenicity of the cells irradiated in different pH medium was determined, and the progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry.

: Irradiation with 2–12 Gy of γ-rats induced apoptosis in SCK cells in pH 7.5 medium within 48 h as judged from the results of four different assays mentioned. Radiation-induced apoptosis declined as the medium pH was lowered from 7.5 to 6.4. Specifically, the radiation-induced degradation of DNA including the early DNA breaks, as determined with the TUNEL method, progressively declined as the medium pH was lowered so that little DNA fragmentation occurred 48 h after irradiation with 12 Gy in pH 6.6 medium. When the cells were irradiated and incubated for 48 h in pH 6.6 medium and the medium was replaced with pH 7.5 medium, DNA fragmentation promptly occurred. DNA fragmentation also occurred even in pH 6.6 medium when the cells were irradiated and maintained in pH 7.5 medium for 8 or longer post-irradiation before incubation in pH 6.6 medium. The radiation-induced G₂ arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium.

: Radiation-induced apoptosis in SCK cells in vitro is reversibly suppressed in an acidic environment. Taking the results of four different assays together, it was concluded that early step(s) in the apoptotic pathway, probably the DNA break or upstream of DNA break, is reversibly halted by an acidic environment in irradiated cells. Radiation-induced G₂ arrest is prolonged in an acidic environment indicating that the suppression of radiation-induced apoptosis and prolongation of radiation-induced G₂ arrest in an acidic environment are related.     

抑制蛋白磷酸酶2A对鼻咽癌放射敏感性的影响

目的：研究放射处理条件下蛋白磷酸酶2A （PP2A）的抑制对鼻咽癌CNE2细胞及其裸鼠移植瘤放射增敏的效果。方法：建立鼻咽癌细胞CNE2体外实验与裸鼠体内移植瘤模型,随机分为空白对照组（PBS）、单纯去甲斑蝥素药物组（NCTD,体外40μmol/L,体内27μmol/kg）、单纯放射组（体外8 Gy,体内20 Gy）及联合处理组。采用四甲基噻唑蓝（MTT）、免疫共沉淀、流式细胞术等方法研究去甲斑蝥素和或放射处理对鼻咽癌CNE2细胞PP2A活性、细胞周期、细胞凋亡及裸鼠体内移植瘤的影响。结果：体外和体内实验均显示,单纯NCTD处理组5 h后PP2A活性均被抑制,约为对照组的70%,而单纯放射组处理5 h后PP2A蛋白活性明显升高,分别约为对照组的210%（体外）和165%（体内）,差异均具有统计学意义（P<0.05）。单纯NCTD处理组、单纯放射组均可不同程度的引起CNE2细胞G2/M期阻滞及细胞凋亡率的增加,与对照组间的差异均具有统计学意义（P<0.05）。联合处理组与对照组比较,PP2A的活性明显降低,分别约为对照组的80%（体外细胞实验）和72%（体内裸鼠实验）;同时G2/M期细胞显著增多（87.88%±2.10%）,细胞凋亡率也明显提高（77.15%±7.62%）;裸鼠移植瘤抑瘤率达到87.98%,其差异均具有统计学意义（P均<0.05）。移植瘤组织病理学观察发现联合处理组大量细胞坏死,部分融合成片,核膜核仁破碎,在肿瘤组织细胞非死亡区可见到被瘤细胞吞噬形成的凋亡小体。结论：PP2A很有可能成为提高鼻咽癌临床放射治疗作用的增敏新靶点。     

BCR/ABL Oncoprotein-Targeted Antitumor Activity of Antisense Oligodeoxynucleotides Complementary to BCR/ABL mRNA and Herbimycin A, an Antagonist of Protein Tyrosine Kinase: Inhibitory Effects on In Vitro Growth of Ph1-Positive Leukemia Cells and BCR/ABL Oncoprotein-Associated Transformed Cells

We investigated whether antisense oligodeoxynucleotides complementary to bcr/abl mRNA or protein kinase antagonists display antitumor activity on Ph -positive leukemia cell lines. bcr/abl antisense oligomers showed inhibitory effects on the in vitro growth of Ph¹-positive leukemia cell lines in liquid culture, and further displayed an inhibitory effect on transformed murine hematopoietic cells using transfection with a retroviral vector expressing p210^bcr/abl oncoprotein. However, in vitro treatment with a bcr/abl antisense oligomer did not completely abolish the expression of bcr/abl mRNA and did not display the desired “killing effect” on Ph¹-positive leukemia cells. On the other hand, investigation of the effect on Ph¹-positive leukemia cells by various types of protein kinase antagonists revealed that herbimycin A, a protein tyrosine kinase antagonist, displays preferential and remarkable suppression of the growth of Ph¹-positive leukemia cells and P210^bcr/abl associated transformed cells by virtue of suppressing bcr/abl protein tyrosine kinase activity. These results may provide important future insights in developing a new category of antitumor therapy by targeting oncogene products.     

The sensitivity of chronic lymphocytic leukaemia lymphocytes to irradiation in vitro

The inhibition of [³H]-thymidine incorporation into the DMA of mitogen-stimulated chronic lymphocytic leukaemia lymphocytes by chlorambucil or γ-irradiation in vitro was measured in a series of patients, some of whom were untreated, some treated and some who were showing resistance to first-line or second-line treatment. There was evidence of resistance to irradiation developing in parallel with that to chlorambucil. The resistance to chlorambucil in chronic lymphocytic leukaemia (CLL) is not necessarily due to altered drug transport or metabolism but to a more fundamental process affecting DNA damage.     

枸杞多糖对脊髓神经细胞辐射损伤后的保护作用研究

目的：研究枸杞多糖（LBP）对辐射损伤脊髓神经（SCN）细胞的保护作用,并观察自噬相关蛋白LC3 Ⅱ/I表达的变化,探讨LBP对辐射损伤保护作用的机制。方法：体外培养SCN细胞,应用不同剂量（0、2、6、10 Gy）X射线辐照损伤后,采用四甲基噻唑蓝（MTT）法检测细胞存活率确定最佳辐射剂量,建立辐射损伤模型。用不同浓度（15、25、40 mg/L）LBP对辐射损伤的SCN细胞进行干预后,分别采用MTT法检测细胞存活率以确定最适LBP干预剂量;用最佳辐射剂量和LBP干预后,采用Western blot和免疫组化法检测自噬相关蛋白LC3 Ⅱ/I的表达。结果：X射线辐照后,SCN细胞存活率在2~10 Gy范围内随着照射剂量的增加而逐渐降低,和正常对照组比较差异均具有统计学意义（P < 0.05）;MTT实验结果显示,与辐射组相比,不同浓度LBP干预后SCN细胞存活率均明显升高,差异具有统计学意义（P < 0.05）,故确定10 Gy X射线照射和40 mg/L LBP干预进行后续造模。免疫组化和Western blot检测结果均显示,与正常对照组相比,辐射组细胞的自噬相关蛋白LC3 Ⅱ/I表达明显升高,差异具有统计学意义（P < 0.05）;与辐射组相比,LBP+辐射组细胞LC3 Ⅱ/I蛋白表达亦明显升高,差异具有统计学意义（P < 0.05）。结论：LBP对体外培养的脊髓神经元辐射损伤具有保护作用,可能与LBP促进自噬相关蛋白LC3 Ⅱ/I表达有关。     

多溴联苯醚BDE-3、BDE-47和BDE-209的体外遗传毒性评价

目的：对3种代表性多溴联苯醚（PBDEs）BDE-3、BDE-47和BDE-209进行体外遗传毒性评价。方法：采用TK6人淋巴母细胞进行彗星试验、微核试验和TK基因突变试验,同时检测DNA损伤、染色体改变和基因突变3个遗传学终点。每种PBDEs均设定5个剂量组：BDE-3为60、90、120、180和240 μmol/L;BDE-47为60、120、180、200和240 μmol/L;BDE-209为24、40、120、180和240 μmol/L;同时设定溶媒DMSO为阴性对照组。结果：与阴性对照组比较,3种PBDEs在各个剂量下均未能引起TK6细胞彗星的尾长、尾部DNA百分数和尾矩的增加（P > 0.05）,也均未引起TK6细胞微核率升高（P > 0.05）。TK基因突变试验显示,3种PBDEs均能引起TK基因突变频率升高,差异具有统计学意义（P < 0.05）,并呈剂量依赖性升高趋势（相关系数[R_BDE-3²]=0.85,[R_BDE-47²]=0.85,[R_BDE-209²]=0.90;P < 0.05）,但致突变作用BDE-47强于BDE-3和BDE-209。结论：多溴联苯醚BDE-3、BDE-47和BDE-209对TK6细胞具有致突变作用。     

Activation of Bcr-abl Fusion Gene and Ras Oncogenes in Chronic Myelogenous Leukemia

We attempted to detect the bcr-abl fusion gene and ras gene family in CML by the in vitro focus forming assay and the tumorigenicity assay. Eight of 14 chronic phase and both of two blastic phase cases showed transforming activity in the tumorigenicity assay. However, only one chronic phase sample was positive in the in vitro focus forming assay. Among these 10 transformants, we found N-ras activation in one chronic phase, and K-ras activation in another chronic phase case. The bcr-abl fusion gene was activated in one chronic phase and all of the blastic phase cases by the tumorigenicity assay. The present result showed that the bcr-abl fusion gene transfected N1H3T3 cells formed tumors in nude mice in contrast to the in vitro focus forming assay. The bcr-abl fusion gene may play important roles in the progression as well as the pathogenesis of CML.     

Dual-isotope lymphoscintigraphy using albumin nanocolloid differentially labeled with 111In and 99mTc

The aim of this study was to develop and evaluate ¹¹¹In- and ^99mTc-labeled derivatives of albumin nanocolloid (NC) for dual-label lymphoscintigraphy to allow simultaneous comparison of lymphatic flow from different tissue planes draining a tumour bed for accurate identification of sentinel lymph nodes (SLN). Using the chelator, p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (DOTA), ¹¹¹In-DOTA-NC and ^99mTc-DOTA-NC were compared in vitro with respect to stability of labeling, colloidal status and particle size, then in vivo by measuring their clearance rates from a subcutaneous injection depot. ¹¹¹In-DOTA-NC and ^99mTc-DOTA-NC were indistinguishable on the basis of in vitro criteria. Their in vivo clearance rates, however, were disparate (0.0015 to 0.075 min^-1 for ¹¹¹In and 0.0072 to 0.067 min^-1 for ^99mTc), ¹¹¹In being faster in three studies and markedly slower in three. This demonstrates that even when dual-labeled radiotracers behave identically in vitro, they will not necessarily do so in vivo. Further work is needed to develop dual-labeled NC.     

PM2.5对支气管上皮细胞DNA损伤作用研究

目的：探讨大气细颗粒物（PM_2.5）染毒对人支气管上皮细胞（HBE） DNA损伤的作用。方法：分别用8、20、50 μg/mL的PM_2.5水溶液染毒HBE细胞24 h后,单细胞凝胶电泳实验（SCGE）检测DNA损伤情况。10和50 μg/L的PM_2.5水溶液染毒HBE细胞,以未染毒细胞作为阴性对照组,10 μmol/L的Cr⁶⁺水溶液为阳性对照组,实时荧光定量PCR （qPCR）检测DNA损伤修复基因hOGG1、hMTH1的mRNA表达水平的变化,Western blot检测hOGG1、hMTH1蛋白表达变化。结果：单细胞凝胶电泳检测8、20和50 μg/mL PM_2.5水溶液染毒组HBE细胞的尾部DNA含量、尾长、尾距较阴性对照组明显增加（P < 0.05或P < 0.01）。qPCR结果显示,与阴性对照组比较,HBE细胞hOGG1 mRNA表达水平在10和50 μg/mL PM_2.5水溶液染毒以及阳性对照Cr⁶⁺水溶液染毒后分别升高75.0%、132.0%、214.0%;hMTH1 mRNA分别升高61.0%、144.0%、75.0%。Western blot结果显示,与阴性对照组比较,HBE细胞hOGG1蛋白表达水平在10和50 μg/mL PM_2.5水溶液染毒以及Cr⁶⁺水溶液染毒后分别升高47.6%、64.0%、47.0%;hMTH1蛋白分别升高20.5%、49.8%、20.9%。结论：PM_2.5水溶液染毒对HBE细胞DNA具有明显的损伤作用,并引起HBE细胞DNA损伤修复基因hOGG1、hMTH1表达水平升高。     

Overexpression of the 27 KDa heat shock protein is associated with thermoresistance and chemoresistance but not with radioresistance

Purpose: The heat shock protein (hsp27) correlates with thermotolerance and chemoresistance. Our main objective was to assess the response to radiotherapy both in vitro and in vivo in correlation with various concentrations of hsp27. The second objective was to evaluate the relation between hsp27 and glutathione-s-transferase π (GST π).

Methods and Materials: For the in vitro study, thermoresistant cell lines, expressing various amounts of hsp27, were used to assess the role of this protein in radioresistance. To verify the efficiency of hsp27 in these cells lines to confer resistance to cytotoxic agents, these cells were also treated with heat shock and cisplatin. Furthermore, the role of hsp27 expression was studied in vivo by immunochemistry in 98 patients with head and neck squamous cell carcinoma treated by radiotherapy. hsp27 was correlated with local control of the tumor and with clinical and biologic factors potentially able to affect the local control, including p53, ki-67, ploidy, and GST.

Results: In vitro, high constitutive levels of expression of hsp27 did not significantly influence the survival curves of transfected cells exposed to radiation as compared to control cells although hsp27 overexpression was confirmed to increased the cellular resistance to heat and to cisplatinum. In vivo, we showed that overexpression of various amounts of hsp27 did not correlate with local control of the tumor. In vivo, hsp27 was only significantly associated with GST π. Expression of GST π was associated with poor local (p = 0.01) control and survival (p = 0.08) in a Cox model.

Conclusions: It is concluded that the mechanisms responsible for hsp27-mediated heat and drug resistance are not involved in radioprotection.     

No preferential sensitivity of t(8;21) acute myeloid leukemias to cytosine arabinoside in vitro: is intensity of therapy or high dose Ara-C crucial for response?

Acute myeloid leukemia (AML) patients with core binding factor abnormalities [inv(16) or t(8;21)] have a relatively good prognosis, especially patients with inv(16) when treated with high-dose cytosine arabinoside (AraC) containing regimens, whereas in the case of t(8;21) evidences in favor of such regimen are contrasting. We previously demonstrated that blast cells from inv(16)-positive AML patients are characterized by an increased sensitivity to AraC with higher incorporation of ³H AraC into DNA and the increase of induced apoptosis in vitro. In the present study we tested the sensitivity of leukemic cells from 15 t(8;21)-positive AML patients to AraC and compared it with the results obtained from cells of 74 patients with inv(16), “intermediate” or “unfavourable” karyotype at diagnosis (for a total of 89 patients). The incorporation of ³H AraC into DNA in cells with t(8;21) was significantly lower than in cells with inv(16) (P = 0.02) or normal karyotype (P = 0.04). Interestingly, the incorporation of the drug into DNA in t(8;21) cells was similar to those with “unfavourable” karyotype. Furthermore, AraC induced apoptosis in t(8;21)-positive AML cells was not increased. These data suggest that the mechanism of response to chemotherapy for t(8;21)-positive cells is probably different then in AML cells with inv(16), underlining the possible importance for patients carrying the t(8;21) of repeated high-dose regimens and not necessarily of high-dose AraC based ones.     

Comparison of in vitro and in vivo effects of vincristine and vindesine on leukemic cells from patients with chronic granulocytic leukemia in blast crisis

We employed a liquid culture system to examine the in vitro effects of vincristine and vindesine on cellular incorporation of ³⁵S0₄ into leukemic cells obtained from 5 patients with chronic granulocytic leukemia in blast crisis. The per cent of ³⁵S0₄ into drug-treated as compared to saline-treated leukemic cells was compared to the clinical outcome of patients treated with these agents. A good or partial clinical response to vincristine or vindesine was seen in patients whose leukemic cells incorporated less than 50% ³⁵S0₄ when exposed to vincristine or vindesine in vitro compared with control saline-treated cells. No clinical response was observed following treatment with vincristine or vindesine if the ³⁵S0₄ incorporation of drug treated leukemic cells was greater than 50% of saline-treated cells. These data suggest that the in vitro effects of vincristine or vindesine on ³⁵S0₄ incorporation into leukemic cells of patients in blast crisis may parallel the clinical outcome of patients treated with these agents in vivo     

Effects of recombinant human granulocyte and granulocyte-macrophage colony-stimulating factors on neutrophil function following autologous bone marrow transplantation

Functional activity of peripheral blood neutrophils was assessed in eight patients at 4, 6, 8, 10 and 12 weeks following autologous bone marrow transplantation (ABMT). Functions studied included superoxide generation (O₂⁻) intracellular killing of Staphylococcus aureus, phagocytosis and killing of Candida albicans. Neutrophils were tested following in vitro preincubation with 300 pM granulocyte-macrophage colony-stimulating factor (GM-CSF), 1.2 nM granulocyte colony-stimulating factor (G-CSF) or buffered solution (diluent) as control. Our data indicate that during the early period (weeks 4–6) following ABMT most of the patients exhibited diminished neutrophil oxidative metabolism, defective phagocytosis and killing of C. albicans and reduced capacity to kill S. aureus. In some patients a gradual increase in the functional activity of neutrophils occurred with time. Both GM-CSF and G-CSF induced in vitro amplification of (a) O₂⁻ production in response to fmet-leu-phe (FMLP) (b) phagocytosis and killing of C. albicans and (c) killing of S. aureus. This study suggests that GM-CSF and G-CSF may enhance the depressed functional activity of neutrophils following ABMT.     

DNA supercoiling changes and nuclear matrix-associated proteins: Possible role in oncogene-mediated radioresistance

: Transfection with either H-ras or H-ras and c-myc has been shown to confer radioresistance in rat embryonal cells (REC). REC primary, transfected with either c-myc or cotransfected with c-myc and H-ras (in ascending order of radioresistance and tumorigenicity), were used as an in vitro model system to determine if nuclear matrix-mediated higher order DNA organization contributes to oncogene-mediated radioresistance.

: DNA damage induction and repair were measured by the alkaline and neutral filter elution assays. Analysis of the ability of DNA loop domains to undergo supercoiling changes in the presence of radiation-induced damage was determined by the fluorescent halo assay 9FHA). Because DNA loops are organized by the nuclear matrix (NM), a study of NM-associated proteins by high resolution two0dimensional gel electrophoresis was performed.

Results: Induction and repair rates of DNA single- and double-strand breaks were similar for the relatively radiosensitive c-myc transfected and the radioresistant c-myc + H-ras transfected cells. However, the degree of inhibition of DNA supercoil rewinding in the presence of radiation-induced damage was less in the radioresistant cells and was inversely correlated with survival. A. progressive loss of NM-associated proteins was observed, which correlated with increasing radioresistance and tumorigenicity in these cell lines. In addition, some protein changes were consistent with the possibility that these changes could be involved in DNA anchoring.

: Increased radioresistance associated with increasing tumorigenicity in these oncogene-transfected cell lines could be due to changes in NM-mediated DNA organization, possibly via differences in NM protein composition that occur following oncogenic transfection.     

Pre-natal, clonal origin of acute lymphoblastic leukaemia in triplets

A unique case of ALL in three monozygotic triplets diagnosed at the age of 24, 27 and 37 months is described. Archived bone marrow smears were available for molecular analysis of immunoglobulin heavy chain (IGH) and IGκ genes and T-cell receptor (TCR)-δ and γ gene rearrangements. A shared IGH rearrangement was found in triplets “A” and “B”, and an identical rearrangement of TCR-δ in triplets “B” and “C”. These data suggest a common, monoclonal initiation of ALL in one of these three triplets, followed by dissemination of clonal progeny to the other twins via vascular anastomoses within the single, monochorionic placenta that they shared in utero. Differences in IGH rearrangements in diagnostic samples also indicates divergent subclonal evolution of the original “pre-leukaemic” clone.     

In vitro hormonal responsiveness of human blood and bone marrow cells in non-lymphocytic leukemia

Glucocorticoid, β-adrenergic agents and prostaglandin E₂ are all known to modulate the proliferation and differentiation of granulocyte-monocyte colonies (CFU-C). However, in man, acute non lymphoid leukemia (ANLL) has been shown to respond in a highly variable manner to glucocorticoid therapy. Therefore, we have measured in vitro on blood and bone-marrow cells of two patients with ANLL several parameters including glucocorticoid receptors, percentage of cells in the S phase of the cell-cycle, effects of drugs on nucleoside incorporation, cell number and viability.

Our results led to the following conclusions: (i) the examination of blood and bone marrow samples in a given patient does not necessarily give similar results in terms of response to drugs and levels of glucocorticoid receptors, (ii) in the same patient, the effect of any agent varies greatly throughout the incubation period and is also variable from one cell subpopulation to another, (iii) prostaglandin E₂ markedly enhances the level of [³H]-thymidine incorporation in the two patients tested. As steroid-induced prostaglandin production has been recently implicated in the differentiation of myeloid cells, this may explain the stimulatory action of glucocorticoids demonstrated both in vivo and in vitro in ANLL patients.     

DNA strand breaks in human leukocytes induced by chemotherapy and total body irradiation

The occurrence of DNA strand breaks and/or DNA alkali-labile sites in peripheral blood leucocytes was demonstrated ex vivo in three patients during and after bone marrow ablative chemotherapy and total body irradiation (TBI) with use of fluorometric analysis of the DNA unwinding rate in alkaline solution (FADU assay). DNA damage was apparent after cyclophosphamide administration and after TBI, related to the amount of the applied dose. In vivo repair occurred within 24 hours, although not to pretreatment values. Demethoxydaunorubicin and busulfan at the dosages used did not induce measurable DNA strand breaks. The experiences described may be developed further to study ex vivo the occurrence of DNA lesions in patients during and after anticancer treatment. Such studies may be of value in comparing the DNA damaging potential of different chemotherapeutic or radiotherapeutic regimens and as a biological assessment of DNA damage after nuclear casualties in cases where the dose is greater than 1-2 Gy and measurement can be made within due time after the ionizing exposure.