Hyper-activation of Aurora kinase a-polo-like kinase 1-FOXM1 axis promotes chronic myeloid leukemia resistance to tyrosine kinase inhibitors.

BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL1 fusion protein. Accordingly, TK inhibitors have drastically changed the disease prognosis. However, persistence of the transformed hematopoiesis even in patients who achieved a complete response to TK inhibitors and the disease relapse upon therapy discontinuation represent a major obstacle to CML cure. METHODS: Thiostrepton, Danusertib and Volasertib were used to investigate the effects of FOXM1, AKA and Plk1 inhibition in K562-S and K562-R cells. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Quantitative reverse transcription (RT)-PCR was used to assess BCR-ABL1, FOXM1, PLK1 and AURKA expression. Protein expression and activation was assessed by Western Blotting (WB). Clonogenic assay were performed to confirm K562-R resistance to Imatinib and to evaluate cells sensitivity to the different drugs. RESULTS: Here we proved that BCR-ABL1 TK-dependent hyper-activation of Aurora kinase A (AURKA)-Polo-like kinase 1 (PLK1)-FOXM1 axis is associated with the outcome of Imatinib (IM) resistance in an experimental model (K562 cell line) and bone marrow hematopoietic cells. Notably, such a biomolecular trait was detected in the putative leukemic stem cell (LSC) compartment characterized by a CD34+ phenotype. Constitutive phosphorylation of FOXM1 associated with BCR-ABL1 TK lets FOXM1 binding with ß-catenin enables ß-catenin nuclear import and recruitment to T cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription complex, hence supporting leukemic cell proliferation and survival. Lastly, the inhibition of single components of AURKA-PLK1-FOXM1 axis in response to specific drugs raises the expression of growth factor/DNA damage-inducible gene a (GADD45a), a strong inhibitor of AURKA and, as so, a critical component whose induction may mediate the eradication of leukemic clone. CONCLUSIONS: Our conclusion is that AURKA, PLK1 and FOXM1 inhibition may be considered as a promising therapeutic approach to cure CML.

Modulation of apoptotic signalling by 9-hydroxystearic acid in osteosarcoma cells.

9-hydroxystearic acid (9-HSA) belongs to the class of endogenous lipid peroxidation by-products that greatly diminish in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity. In this paper our attention has not only been focused on HDAC1 inhibition but also on the hyperacetylation of other substrates such as p53, that is involved in inducing cell cycle arrest and/or apoptosis, and whose activity and stability are known to be regulated by posttranslational modifications, particularly by acetylation at the C-terminus region. 9-HSA administration to U2OS, an osteosarcoma cell line p53 wt, induces a growth arrest of the cells in G2/M and apoptosis via a mitochondrial pathway. In particular hyperacetylation of p53 induced by the HDAC1 inhibitory activity of 9-HSA has been demonstrated to increase Bax synthesis both at the transcriptional and the translational level. The subsequent translocation of Bax to the mitochondria is associated to a significant increase in caspase 9 activity. Our data demonstrate that the effects of 9-HSA on U2OS correlate with posttranslational modifications of p53.

Growth patterns, cell composition, and mitotic activity of granulocytic aggregates developing in methyl cellulose from human normal marrow cells.

Marrow cells from five normal healthy subjects were cultured in methyl cellulose with and without leucocyte conditioned medium. Cultures were examined daily from day 3 to day 14 for the number of granulocytic aggregates and for their cell composition and mitotic activity. Aggregates of different size (small and large clusters, small and large colonies) displayed four distinct and characteristic patterns of growth. Unidentifiable blast cells were very rare throughout culture. The average proportion of recognizable granulocyte precursors (promyelocytes and myelocytes) (PMC + MC) declined during culture from 60% to 34%. There was a negative correlation between the size of the aggregates and their PMC + MC content, but on any day of culture some aggregates consisted mainly or uniquely of PMC + MC, while some others consisted mainly or uniquely of non-proliferating granulocytes. The mitotic index of PMC + MC was much higher than the one previously determined in vivo, and was significantly higher in large colonies than in other aggregates. These results are consistent with the recent demonstration of different CFU-C (sub)populations. It is suggested that differentiation of the aggregates according to their size and kinetics, coupled with cytological examination, can provide a more refined assessment of the assay, which could be useful for further comparison of normal, abnormal, and leukaemic granulocytopoiesis.

Mutagenic and toxic effects of 4-hydroperoxycyclophosphamide and of 2,4-tetrahydrocyclohexylamine (ASTA-Z-7557) on human lymphocytes cultured in vitro.

Certain biological effects exerted by 4-hydroperoxycyclophosphamide and by 2,4-tetrahydrocyclohexylamine (ASTA-Z-7557), utilized in vitro in the therapy of leukemias and lymphomas to eliminate the occult tumor cells in autologous marrow transplantations, were studied in human lymphocytes cultured in vitro. The data show that these drugs exert mutagenic activity eliciting unscheduled DNA synthesis (reparative synthesis) after DNA damage and cause about tenfold higher frequency of sister chromatid exchanges than controls. Furthermore, they exert strong toxic effects, measured as tritiated thymidine uptake inhibition, on mitogen-stimulated dividing cells even if pretreated during the nonproliferative phase of the cell cycle in which the toxic activity of the drugs is not detectable. Data obtained with doses of the drugs similar to those used in the therapy are discussed in terms of the therapeutic use of these chemicals.

Long-term bone marrow cultures in Diamond-Blackfan anemia reveal a defect of both granulomacrophage and erythroid progenitors.

The hematopoietic defect of Diamond-Blackfan anemia (DBA) results in selective failure of erythropoiesis. Thus far, it is not known whether this defect originates from an intrinsic impediment of hematopoietic progenitors to move forward along the erythroid pathway or to the impaired capacity of the bone marrow (BM) microenvironment to support proliferation and differentiation of hematopoietic cells. Reduced longevity of long-term bone marrow cultures, the most physiologic in vitro system to study the interactions of hematopoietic progenitors and hematopoietic microenvironment, is consistent with a defect of an early hematopoietic progenitor in DBA. However, stromal adherent layers from DBA patients generated in a long-term culture system, the in vitro counterpart of BM microenvironment, did not show evidence of any morphologic, phenotypic, or functional abnormality. Our major finding was an impaired capacity of enriched CD34+ BM cell fraction from DBA patients, cultured in the presence of normal BM stromal cells, to proliferate and differentiate along the erythroid pathway. A similar impairment was observed in some DBA patients along the granulomacrophage pathway. Our result points to an intrinsic defect of a hematopoietic progenitor with bilineage potential that is earlier than previously suspected as a relevant pathogenetic mechanism of the disease. The finding of impaired granulopoiesis in some DBA patients underlines the heterogeneity of this rare disorder.

Expression of P21(WAF1/CIP1/SID1) cyclin-dependent kinase inhibitor in hematopoietic progenitor cells.

P21(Waf1/Cip1/Sid1) is a critical component of biomolecular pathways leading to the G(1) arrest evoked in response to DNA damage, growth arrest signals and differentiation commitment. It belongs to the Cip/Kip class of cyclin-dependent kinase inhibitors and is at least partly regulated by p53. P21(Waf1/Cip1/Sid1) functional inactivation possibly resulting from mutations of the gene itself or, more likely, from p53 mutations may be critical for either the cell fate following DNA-damaging insults or clonal evolution toward malignancy. In the study presented here we describe a competitive polymerase chain reaction (PCR) strategy whose sensitivity and reproducibility enable us to attain a precise quantitation of p21(Waf1/Cip1/Sid1) expression levels in hematopoietic progenitors, the cell compartment which mostly suffers from the side effects of genotoxic drugs in use for cancer cure. The strategy was set in the M07 factor-dependent hematopoietic progenitor cell line. We confirmed that its p21(waf1/cip1/sid1) constitutive expression level is very low and up-modulated by DNA-damaging agents: ionizing radiations and ultraviolet light. Gene up-modulation resulted in checkpoint activation and, in particular, in a significant G(1) arrest, required for either the repair of damaged DNA sequences or apoptotic cell death. Our competitive PCR strategy was further validated in CD34(+) purified hematopoietic progenitors from healthy donors mobilized into the peripheral blood by granulocyte colony-stimulating factor and intended for allogeneic bone marrow transplantation. The constitutive p21(WAF1/CIP1/SID1) expression levels, measured in three separate harvests, were very low and no significant differences were apparent. Our results support the use of a competitive PCR strategy as a useful tool for clinical purposes, to assess the individual biomolecular response of early hematopoietic progenitors to antiblastic drugs.

The v-abl, c-fms, or v-myc oncogene induces gamma radiation resistance of hematopoietic progenitor cell line 32d cl 3 at clinical low dose rate.

A variety of viral and cellular oncogenes have been described with differing mechanisms of action but with the common property of inducing morphologic alteration of cells in culture. Subclonal lines of oncogene expressing cells have been shown to produce tumors in vivo. Expression of the N-ras oncogene in embryo fibroblast NIH/3T3 cells has been demonstrated to increase radioresistance in vitro, and these results have been confirmed and extended to human cell lines expressing the c-raf oncogene. In the present report, we have examined the effects of expression of the c-fms, v-abl, or v-myc oncogene in a clonal hematopoietic progenitor cell line 32D cl 3. The 32D cell line is nonmalignant in vivo and is dependent upon a source of Interleukin-3 (IL-3) for growth in vitro. The radiation survival of 32D cl 3 cells transfected and expressed in the c-fms oncogene showed significant increase in the radioresistance at both 5 cGy/min and 116 cGy/min. A clone of 32D cl 3 transfected and expressing the v-myc oncogene demonstrated increased radioresistance at both dose rates. Results of split dose experiments suggested significant repair of sublethal irradiation damage of 32D-v-abl cells. Results were compared with expression of the same v-abl oncogene in the NIH/3T3 embryo fibroblast cell line. The data demonstrate that gamma irradiation resistance is significantly increased by each oncogene expressed in 32D cl 3 cells. The data on cell line 32D cl 3 may correlate with the radioresistance of v-abl expressing human hematopoietic cell malignancies treated by irradiation therapy.

Recombinant murine GM-CSF increases resistance of some factor dependent hematopoietic progenitor cells to low-dose-rate gamma irradiation.

The effects of murine recombinant IL-3 (multi-CSF) and murine recombinant GM-CSF (granulocyte-macrophage colony stimulating factor) on the radiation biology of clonal hematopoietic progenitor cell lines were evaluated. Four clonal cell lines with growth response to either IL-3 or GM-CSF (FDCP-1JL26, and bg/bg d64) or exclusively dependent on IL-3 (32D cl 3 and B6SUtA), were pre-incubated in IL-3, or GM-CSF, for 7 days prior to gamma irradiation, then washed and irradiated at 5 cGy/min, or 116 cGy/min, and transferred to semisolid medium supplemented with either IL-3, or GM-CSF, for assay of 7 day greater than or equal to 50 cell colonies. The cell lines demonstrated similar radiosensitivity and lack of a detectable dose-rate effect when grown in IL-3 (FDCP-1JL26: D0 154, n 1.05 at 5 cGy/min, and D0 138, n 1.16 at 116 cGy/min; bg/bg d64: D0 95.7, n 1.16 at 5 cGy/min, and D0 97.7 n .993 at 116 cGy/min; B6SUtA: D0 101, n 1.29 at 5 cGy/min, D0 100, n 1.27 at 116 cGy/min; and cell line 32D cl 3: D0 123, n 1.65 at 5 cGy/min, and D0 126, n 1.17 at 116 cGy/min). In contrast, FDCP-1JL26 cells demonstrated a significant relative radioresistance at low-dose-rate when grown in recombinant GM-CSF, (D0 217, n 1.27 at 5 cGy/min, D0 138, n 1.34 at 116 cGy/min, p less than .005). The increase in radioresistance of FDCP-1 cells at low-dose-rate was induced either by preincubation in GM-CSF with transfer to IL-3, or by preincubation in IL-3 and transfer to recombinant GM-CSF. Growth factor independent malignant subclones of lines B6SUtA and FDCP-1JL26 demonstrated a significant increase in radioresistance at low-dose-rate (B6SUtA EL4JL: D0 187, n 1.39 at 5 cGy/min, and D0 133, n 1.73 at 116 cGy/min (p. less than .05); and FDCP-1JL26 F7 cl 2: D0 191, n 1.17 at 5 cGy/min, and D0 150, n 1.31 at 116 cGy/min [p less than .05]). Thus, some hematopoietic progenitor cell lines are induced by GM-CSF to grow after irradiation at low-dose-rate similar to the growth of clonal malignant cell lines. The data may have implications for the radiation biology of normal hematopoietic progenitor cells in two circumstances: (a) selective survival of GM-CSF responsive cells after total body irradiation, and (b) selective survival of some hematopoietic progenitors in vivo during clinical recombinant GM-CSF infusion.

Radiation-induced gadd45 expression correlates with clinical response to radiotherapy of cervical carcinoma.

BACKGROUND: Recent work has identified a category of genes devoted to the control of genomic stability and prevention of cellular evolution. They encode components of cell cycle checkpoint, i.e., regulatory pathways committed to ordered cell cycle transition and fidelity of replicated DNA under adverse environmental conditions, such as those following exposure to genotoxic agents. Gadd45 belongs to the class II family of DNA damage-inducible (DDI) gene, and its role in DNA repair has been proved in many experimental models. The aim of our study was to correlate gadd45 radio-induction with the responsiveness to radiotherapy of cervical carcinomas, a type of cancer most commonly treated with radiotherapy alone. METHODS: By means of a competitive polymerase chain reaction strategy, we compared in 14 patients the gene expression levels before and during external beam radiotherapy, when a dose ranging from 18 to 25 Gy was delivered to the target. RESULTS: We found a correlation between the lack of gadd45 induction and a good clinical response to radiotherapy, in terms of both local control and disease-free survival. CONCLUSION: Our results support the measure of the induction of gadd45, and possibly of other genes required for regulated G1-S checkpoint, as a method useful for prognostic evaluation of cervical carcinoma patients.

Gamma-irradiation response of cocultivated bone marrow stromal cell lines of differing intrinsic radiosensitivity.

There is evidence for differences in the gamma-irradiation response of different cellular lineages within the bone marrow microenvironment. We previously reported that heterogeneity is demonstrable in the gamma-irradiation response of five clonal stromal cell lines, derived from one human bone marrow specimen, despite morphological, histochemical, cytogenetic, and functional similarity. In the present study we tested whether one stromal cell line could affect the intrinsic radiosensitivity of another. Two clonal stromal cell lines, which display distinct gamma-irradiation responses relative to dose rate were used: KM 101, which shows the same radiosensitivity at a low dose rate of 5 cGy/min (LDR) and a high dose rate of 120 cGy/min (HDR) and KM 104 which shows significant gamma-irradiation resistance at LDR. To facilitate the study of the gamma-irradiation response of each cell line during cocultivation, we derived stable subclones of each, expressing the transfected neomycin resistance (neo-r) gene, which confers resistance to the neomycin analog: G 418. Introduction of the neo-r gene did not alter cell lines radiosensitivity. The results show that cocultivation of stromal cell lines before, during, and after gamma-irradiation induces changes in repair of radiation-induced damage, with a dominant effect of a resistant cell line at LDR. In fact, the radiation survival curves of cocultivated stromal cell lines were always characteristic of KM 104, and a dose rate effect was observed, even when KM 101 was present in large excess. Moreover, our results are consistent with preferential killing of the more radiosensitive stromal cell line: both LDR and HDR Do values of the neo-r KM 101, cocultivated with the parent KM 104 for 24 hr before, and during gamma irradiation were significantly lower compared to the neo-r subclone irradiated alone. The LDR Do value of the neo-r KM 104 cocultivated for 24 hr before, and during gamma irradiation with excess of parent KM 101, was significantly higher, compared to the neo-r cells irradiated alone.

Expression of p210 bcr/abl increases hematopoietic progenitor cell radiosensitivity.

PURPOSE: The cytogenetic finding of the Ph1+ chromosome and its molecular biologic marker bcr/abl gene rearrangement in cells from patients with chronic myeloid leukemia are associated with a proliferative advantage of the Ph1+ clone in vivo. Although the transition to the acute terminal phase or blastic crisis is often associated with additional cytogenetic abnormalities, the molecular events which correlate the initial cytogenetic lesion with the terminal phase are poorly understood. Defective cellular DNA repair capacity is often associated with chromosomal instability, increased mutation frequency, and biologic alterations. METHODS AND MATERIALS: We, therefore, tested whether the protein product of the bcr/abl translocation (p210) could alter DNA repair after gamma-irradiation of murine cell lines expressing the bcr/abl cDNA. RESULTS: The 32D cl 3 parent, 32D cl 3 pYN (containing the control vector plasmid) and each of two sources of 32D cl 3 cells expressing p210 bcr/abl cDNA (32D-PC1 cell line and 32D-LG7 subclone) showed a D0 of 1.62, 1.57, 1.16, and 1.27 Gy, respectively. Thus, expression of the p210 bcr/abl product induced a significant (p < 0.05) increase in radiosensitivity at the clinically relevant radiation therapy dose-rate (1.16 Gy/min). The increased radiosensitivity of p210 bcr/abl expressing cells persisted if cells were held before plating in a density-inhibited state for 8 hr after gamma-irradiation, indicating little effect on the repair of potentially lethal gamma-irradiation damage. The IL-3 dependent parent 32D cl 3 cells demonstrated programmed cell death in the absence of growth factor or following gamma-irradiation to 200 cGy. Expression of bcr/abl cDNA in the 32D-PC1 and 32D-LG7 sub clones abrogated IL-3 requirement of these cell lines and inhibited gamma-irradiation induced programmed cell death. CONCLUSION: These data suggest a role for bcr/abl p210 in amplifying gamma-irradiation DNA damage or broadly inhibiting DNA repair, conditions that may stimulate further cytogenetic alterations in hematopoietic cells.

Radiosensitivity of permanent human bone marrow stromal cell lines: effect of dose rate.

In contrast to the dose-rate independent X ray killing observed with human bone marrow hematopoietic stem cells, bone marrow adherent stromal cells from the same fresh marrow harvests demonstrate increased radiation resistance at low dose rate (LDR) (5 cGy/min), compared to high dose rate (HDR) irradiation (120-200 cGy/min). Physiologic changes observed in plateau phase bone marrow cells after LDR irradiation in vivo and in vitro suggested that marrow stromal cells might be heterogeneous in LDR irradiation repair. Five permanent clonal bone marrow stromal lines were derived from a single human marrow donor. Each cell line was positive for markers of fibroblasts including: immunohistochemically detectable fibronectin, collagen, acid phosphatase, and nonspecific esterase, and was negative for Factor VIII, alkaline phosphatase, lysozyme and several markers of marrow macrophages. The x-irradiation survival curve of each cell line was determined at LDR and HDR in vitro. Cell lines KM102, KM103, KM104, and KM105 each demonstrated a significant (p less than .05) increase in radioresistance at LDR (D0 = 142, n = 2.9; D0 = 131, n = 2.5; D0 = 145, n = 2.1 and D0 = 127, n = 2.1 respectively) compared to HDR: (D0 = 111, n = 2.1; D0 = 94, n = 3.5; D0 = 99, n = 3.5 and D0 = 95, n = 2.1 respectively). In contrast, cell line KM101 demonstrated no significant change in radiosensitivity relative to dose rate at LDR (D0 = 113, n = 3.3) compared to HDR, D0 = 114, n = 3.3. Cell line KM101 was more supportive than the other lines of cocultivated hemopoietic cells in vitro. Subclones of KM101 and KM104 selected by retroviral vector transfer of the neor gene for growth in the antibiotic neomycin-analogue G418, maintained the stably associated radiobiologic properties of each parent clonal line. These data indicate significant heterogeneity in the LDR irradiation response of clonal stromal cell lines derived from human bone marrow.

Radiosensitivity of human prostate cancer and malignant melanoma cell lines.

The relative radioresponsiveness of human prostate cancer compared to malignant melanoma is well known. The effects of beta-estradiol or testosterone on the X-irradiation survival of several human cell lines were studied, including: human prostate carcinoma cell lines PC3 and DU145 and human malignant melanoma cell lines A375 and A875. Lines PC3 and DU145 demonstrated 55-61 fmol per 10(6) cells of androgen receptor with no detectable estrogen or progesterone receptor. Cells were irradiated at 120 cGy/min dose rate. There was no detectable toxicity of up to 10(-4) M testosterone or beta-estradiol on PC3 or DU145 cells in the absence of X-irradiation. At plating efficiencies from 11-13%, and plating densities of 1 x 10(4) cells per 60 cm2 flask, cell lines PC3 and DU145 demonstrated a Do of 108.5 +/- 6.5, n 2.1 +/- 0.7 cGy, and Do of 143.5 +/- 1.5 cGy, n 2.4 +/- 0.5, respectively. The addition of testosterone or beta-estradiol at 10(-4) to 10(-10) M prior to or after, X-irradiation did not alter radiosensitivity. At the same dose rate of 120 cGy/min, malignant melanoma cell lines A375 and A875 had a Do of 125 +/- 2.5 cGy, n 1.56 +/- 0.8 SF2 0.65 +/- 0.03 and line A875 demonstrated a Do of 129 +/- 4.5 cGy, n 1.58 +/- 0.4 SF2 0.55 +/- 0.04, respectively. The radiosensitivity of melanoma cell lines did not decrease at low dose rate 5 cGy/min. Thus, the in vitro radiosensitivity of androgen receptor positive prostate cancer cell lines is not necessarily altered by the presence of androgen before or after irradiation. The data support the concept that all malignant melanoma cell lines do not show a broad-shouldered cell survival curve in vitro and intrinsic cellular radioresistance.

GM-CSF incubation prior to treatment with cytarabine or doxorubicin enhances drug activity against AML cells in vitro: a model for leukemia chemotherapy.

We studied the effect of preincubation with recombinant GM-CSF on the activity of cytarabine and doxorubicin against clonogenic acute myeloid leukemia cells (CFU-AML). Leukemia cells from seven persons with AML, three myeloid cell lines (HL60, KG1, K562) and two control cell lines (U937, MOLT3) were tested. Preincubation with GM-CSF (0.01-0.1 microgram/ml) increased DNA synthesis as measured by tritiated thymidine incorporation and intranuclear Ki67 expression in cells from six persons with AML and in HL60 cells. Leukemia cells preincubated with GM-CSF for 6-48 h were exposed to cytarabine (2-200 micrograms/ml) or doxorubicin (0.01-0.1 microgram/ml) for 3 h and CFU-AML assayed. This approach further reduced CFU-AML in samples from six persons with AML and in HL60 and KG1 cells compared to cells not preincubated with GM-CSF prior to drug treatment. In most instances, reduced CFU-AML correlated with GM-CSF induced DNA synthesis. These data suggest a possible strategy of GM-CSF pretreatment to increase anti-leukemia efficacy of chemotherapy in AML.

Cytogenetic events after bone marrow transplantation for Philadelphia chromosome positive chronic myeloid leukemia.

Allogeneic bone marrow transplantation is the only way to cure patients with Ph1+ chronic myeloid leukemia. It is commonly assumed that, in order to obtain a cure for the patients, the leukemic clone must be completely destroyed by the conditioning treatment and the donor's bone marrow must repopulate the hemopoietic niches leading to a "complete chimera". However, cytogenetic analyses, supported by molecular ones, indicate that Ph1+ cells, far from being completely destroyed by chemo-radiotherapy may persist for a long time, probably in the majority of the patients. As demonstrated by the outcome of patients receiving T-cell depleted marrow, immune mechanisms must be involved in controlling and progressively reducing the size of the residual leukemic clone. Furthermore, immunodulating therapeutic strategies, represented by cyclosporin-A discontinuation or alpha interferon treatment, may successfully reduce the Ph1+ cell population even after a full relapse.

Therapy of advanced acute myeloblastic leukemia with cytarabine and interleukin 2.

A child with acute myelogenous leukemia who relapsed three months after an allogeneic bone marrow transplant received intermediate-dose cytarabine followed by interleukin 2 (IL-2). Complete remission was achieved after the first cycle of IL-2. Five more combined cycles of cytarabine and IL-2 were given over the next year, during which remission has persisted. IL-2 therapy affected serum tumor necrosis factor (TNF), interferon gamma (IFN gamma) and soluble IL-2 receptor (sIL-2r) levels. In vitro cytotoxicity against leukemia cell lines and recipient leukemia cells was also increased.

Host origin of bone marrow fibroblasts following allogeneic bone marrow transplantation for chronic myeloid leukemia.

We investigated the origin of the fibroblastic compartment of stromal hematopoietic microenvironment in eight chronic myeloid leukemia (CML) patients following allogeneic BMT. At the time of the study, all eight CML patients showed complete and long-lasting (14-87 months) engraftment of donor hematopoiesis and absence of clonal Ph-positive hematopoiesis. The study was carried out using in vitro amplification of informative DNA sequences: a Y chromosome specific DNA fragment in three patients who received a sex-mismatched allograft, and locus D1S80, a variable number of tandem repeats polymorphism, in five patients who received a sex-matched allograft. In all cases bone marrow fibroblasts were of recipient origin. These data indicate that with current BMT procedures the stromal compartment of hematopoiesis is not transplantable in humans.

Expression of transfected recombinant oncogenes increases radiation resistance of clonal hematopoietic and fibroblast cell lines selectively at clinical low dose rate.

To determine the effect of oncogene expression on gamma radiation sensitivity of hematopoietic compared to fibroblastic cells, we selected clonal sublines of an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell line 32D cl 3 and NIH/3T3 embryo fibroblastic cells following transfection with each oncogene linked to the mycophenolic acid resistance gene. Each mycophenolic acid-resistant subclone demonstrated high levels of specific poly(A)+ mRNA for each oncogene. The parent line 32D cl 3 demonstrated similar radiosensitivity at 116 cGy/min (D0 126, n 1.17) compared to 5 cGy/min (D0 123, n 1.65). This pattern was not altered in subclones of 32D cl 3 cells transfected with the epidermal growth factor (EGF) receptor gene and grown in EGF (at 116 cGy/min D0 104, n 0.998, at 5 cGy/min D0 115, n 1.09), or in 32D cl 3 cells expressing the v-sis oncogene (at 116 cGy/min D0 122.4, n 1.79, at 5 cGy/min D0 135, n 1.43). In contrast, expression of the transfected oncogenes v-erb-B, v-abl, or v-src conferred significant radioresistance at 5 cGy/min dose rate (D0 194, n 1.77; D0 165.5, n 1.56; D0 171, n 1.28, respectively). With the exception of v-sis, oncogene expression resulted in nonautocrine factor independence of 32D cl 3 subclones, and production of donor origin tumors in syngeneic new-born or adult mice. Two rare spontaneous factor-independent subclones of 32D cl 3 were also tested. Nonautocrine clone 32D cl 2 demonstrated significantly increased radioresistance at low dose rate (D0 186, n 1.63), while autocrine (IL-3 producing) subclone 32D cl 4 revealed no significant increase in radioresistance at 5 cGy/min. The parent fibroblast cell line NIH/3T3 showed an intrinsic relative radioresistance at low dose rate (at 5 cGy/min D0 157.3, n 1.81, compared to 116 cGy/min D0 134.3, n 1.57). Expression in NIH/3T3 of transfected oncogenes v-abl, v-fms, v-fos, or H-ras increased radioresistance at low dose rate (D0 208.6, n 1.61; D0 206.6, n 1.51; D0 167.5, n 1.85; and D0 206.8, n 1.08, respectively). Thus expression of each of several oncogenes induces resistance to gamma irradiation at 5 cGy/min in hematopoietic and fibroblast cell lines. These data may help explain the clinical recurrence of oncogene-expressing leukemia and lymphoma cells after marrow stem cell ablative doses of low-dose-rate total-body irradiation.

The v-src oncogene may not be responsible for the increased radioresistance of hematopoietic progenitor cells expressing v-src.

Infection of the IL-3-dependent, myeloid progenitor cell line 32D cl 3 with murine retroviruses that contain either the wild-type or a temperature-sensitive mutant v-src can render these cells growth-factor independent. These cells also became resistant to gamma irradiation administered at the low-dose rate of 0.05 Gy/min, which is used clinically. The v-src-dependent nature of resistance to gamma irradiation was examined by studying four clones of 32D cl 3 cells that had been infected with a retrovirus carrying the tsLA31A mutant of v-src. The tyrosine-specific kinase activity of this mutant is dramatically reduced at the nonpermissive temperature of 39 degrees C. Cells transformed by v-src and grown at either 34 or 39 degrees C, in the presence or absence of IL-3, demonstrated a significantly higher D0 compared to parental cells examined under identical conditions. In addition, expression of v-src abrogated the synergistic killing effect of heat and gamma irradiation. The D0 of parental 32D cl 3 cells kept at 39 degrees C after gamma irradiation was reduced significantly compared to the D0 of these cells kept at 34 degrees C. This contrasts with data from 32D cl 3 cells infected with either the wild-type v-src or the temperature-sensitive mutant, neither exhibited a synergistic effect in the D0 at either 34 or 39 degrees C. Therefore, while continuous expression of a v-src gene product is required for maintenance of the growth-factor-independent state, v-src does not appear to be responsible for the increased gamma-radiation resistance of these cells at low dose rate.

Can Long-Term Bone Marrow Culture Eliminate Leukemia Cells?

In chronic myeloid leukemia (CML) and acute myeloblastic leukemia (AML), experimental studies have shown growth advantage of the residual normal progenitor cells under long-term culture conditions. In some instances, this culture system has been used as a tool for purging bone marrow, before autotransplant. In this review, we discuss the efficiency of this technique, with respect to the persistence of sufficient numbers of normal stem and/or progenitor cells, capable of restoring hemopoiesis following pre-transplant conditioning regimens, and in relation to the elimination of leukemia cells, capable of causing relapse. Both issues are complex, and mostly still obscure. However if one considers the clinical outcome of patients receiving unpurged autografts, it is unlikely that long-term bone marrow cultures (LTBMCs) will be of substantial benefit in reducing leukemia relapse. Furthermore the clinical efficacy of this technique will in our opinion be difficult, if not impossible, to prove.