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The DNA-dependent protein kinase (DNA-PK) complex plays a crucial role in radiation-induced DNA damage recognition. The complex includes the ku heterodimer, which comprises ku 70 and ku 80 subunits, that binds DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKcs. The activation of the DNA-PK complex was studied in X-irradiated peripheral blood mononuclear cells (PBMC) from subjects of different ages. Radiation-induced changes in the DNA-binding activity of the ku heterodimer, and in the concentrations of ku 70, ku 80, DNA-PKcs and phosphorylated ku 80 were determined in nuclear and cytoplasmic extracts. DNA-binding activity was increased by irradiation only in the nuclear extract of PBMC from young, but not from elderly subjects, whereas it was found unchanged in cytoplasmic extracts regardless of age. The radiation-induced activation of the DNA-PK complex may result from the increased concentrations of ku 80 and DNA-PKcs in the cytoplasm of PBMC from young, but not from elderly subjects, leading to a higher concentration of phosphorylated ku 80 which readily migrates to the nucleus where, after dimerization with ku 70, binds to DNA breaks. These findings suggest major steps involved in DNA-PK activation, and the intracellular and molecular changes that may account for the age-dependent impairment of DNA repair capacity in irradiated mammalian cells.

The DNA-dependent protein kinase (DNA-PK) complex plays a crucial role in radiation-induced DNA damage recognition. The complex includes the ku heterodimer, which comprises ku 70 and ku 80 subunits, that binds DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKCS: The activation of the DNA-PK complex was studied in X-irradiated peripheral blood mononuclear cells (PBMC) from subjects of different ages. Radiation-induced changes in the DNA-binding activity of the ku heterodimer, and in the concentrations of ku 70, ku 80, DNA-PKcs and phosphorylated ku 80 were determined in nuclear and cytoplasmic extracts. DNA-binding activity was increased by irradiation only in the nuclear extract of PBMC from young, but not from elderly subjects, whereas it was found unchanged in cytoplasmic extracts regardless of age. The radiation-induced activation of the DNA-PK complex may result from the increased concentrations of ku 80 and DNA-PKcs in the cytoplasm of PBMC from young, but not from elderly subjects, leading to a higher concentration of phosphorylated ku 80 which readily migrates to the nucleus where, after dimerization with ku 70, binds to DNA breaks. These findings suggest major steps involved in DNA-PK activation, and the intracellular and molecular changes that may account for the age-dependent impairment of DNA repair capacity in irradiated mammalian cells.

Background The aim of this study was to find out if Cobe Trima, a blood cell separator that automatically collects RBC, PLT and plasma, is adequate for routine multiple blood donation by apheresis.Materials and Methods Eighty donors underwent multiple blood component donations by Cobe Trima. Blood counts were determined on the apheresis products to analyze their quality.Results Eighty procedures were performed collecting 193 products. The average platelet yield was 3.5×1011(± 0.46) in the 54 single product (SP) procedures and 7×1011(± 0.88) in the 26 double product (DP) procedures. WBC contamination of the PLT products was 1.7 × 105(1.2–4.2). The mean platelet efficiency was 60 ± 8.35% for SP and 66 ± 9.59% for DP. The hemoglobin (Hb) content per unit was 46.21 g (± 7.84) in 8 DP and 40.82 g (± 6.41) in 34 SP procedures.Conclusion The production of standardized blood components with good PLT yield and low WBC contamination plus high efficiency makes Trima one of the best blood cell separators of the new generation.

This study evaluates stem cell collection procedures performed with the Dideco Excel blood cell separator, with particular attention given to yields and separator collection efficiencies. Patients’ blood precounts and yield parameters related to the harvest capacity of the collection system were investigated. Fifty-five collection procedures were analyzed in 32 patients suffering from hematological malignancies and solid tumors and mobilized with chemotherapy plus G-CSF. The median blood volume processed in each procedure was 15.8 liters (12–19.750), with a blood flow rate of 70 ml/min. Patients had the following median blood precount value: NC 7.81×109/L, CD34+ cells 49.08×103/ml. Leukapheresis procedures gave the following yields: NC 14.95×109, MNC 10.83×109, CD34+ cells 4.37×106; yields/kg, NC 0.21×109kg, MNC 0.15×109/kg CD34+ cells 4.26×106/kg. Procedures show the following collection efficiencies: NC 10.79%, MNC 29.06%, CD34+ 42.33%, PLT 26.5%. The RBC (red blood cell) contamination of the product was (median value) 20.9 ml for each procedure, and for platelets 1.76×1011per procedure. The CD34+ cell precounts strongly correlated with the CD34+ yields/kg (r=0.82. p=0.000). Furthermore the NC and MNC precounts correlated with the CD34+ yields/kg but only the MNC precount correlation is notable (r=0.57, p=0.000). The logistic regression analysis shows that CD34+ (p=0.008) but not NC (po=0.14), MNC (p=0.09), or PLT (p=0.53) precounts significantly influenced the collection of a sufficient dose of CD34+ cells for transplantation (≥ 2.5×106/kg). Eleven of the thirty-two patients have been transplanted till now, and all had a prompt and lasting trilineage engraftment NC >1×109/L on day 12 (10–17). Our data show that the collection system analyzed in this report is able to collect large amounts of progenitor cells, harvesting ≥2.5×106/kg CD34+ cells with a single procedure in 68.8% of patients and assuring complete recovery after stem cell transplantation.

From January 1996 until now, thirty-eight PBSC procedures were carried out on 20 patients suffering from NHL, mobilized by polichemotherapy regimens plus recombinant human Granulocyte-Growth Factor (rhG-CSF). Patients were enrolled in PBSC procedures using Dideco Excel (group A) and Cobe Spectra v.4.7 (group B) blood cell separators. Twelve patients were enrolled in group A (6 males and 6 females, median age 33) and 9 patients in group B (5 males and 4 females, median age 55). The mean White Blood Cell (WBC) and Mononuclear Cells Fraction (MNC) peripheral blood counts were not statistically different in either group and neither were blood CD34+ cell peripheral counts. CD34+ cell peripheral value was predictive of the CD34+ yield while mean values of harvested CD34+ cells were not significantly different. CD34+ cell efficiences were statistically the same. The CD34+ cell purity of the apheresis harvest was statistically different between the two groups (group A = 3.0 ± 2.2%; group B = 1 ± 0.9%) p = 0.001. High CD34+ cell yields were observed in both groups which confirms that both blood cell separators are able to harvest hematopoietic progenitor cells from peripheral blood.

In this work we evaluated the efficacy of stem cell collection with Large Volume Procedures. (LVP), and analysed the importance of the CD34+ cell precount in promoting the collection of a sufficient number of CD34+ cells for transplantation, using the Univariate Logistic Regression analysis. Eighty-nine leukapheresis were performed in 49 patients with hematological malignancies and solid tumors, mobilized with chemotherapy plus Granulocyte Colony Stimulating Factor (G-CSF). For each procedure 15.8 liters of blood were processed. The median value of Nucleated Cells (NC) and CD34+ cells precount was respectively 8.29 × 109/ml (range 1.13÷45.4) and 43.08 × 103/ml (range 1.06÷795.2). Results show the capability of LVP to collect large quantities of hemopoietic progenitors with a median CD34+ cell total yield of 215.02 × 106(range 5.03÷2210). The yields per patients’ body weight were: CD34+ cells 3.23 × 106/kg (range 0.081÷41.58). The regression analysis between blood cell precounts and collection yields gave the following correlations: the CD34+ cell precount correlates with CD34+ yield (r = 0.78 p < 0.00) and with CD34+ cell yield/kg (r = 0.76 p < 0.00). The number of CD34+ cells processed correlated with the number of CD34+ cells collected/kg (r = 0.83 p < 0.000). To investigate the importance of CD 34+ cell precount in promoting CD34+ cell yields ≥2.5 × 106/kg we performed a Univariate Logistic Regression analysis that showed in our patients a probability of collecting ≥2.5 × 106CD34+/kg that rose from 0.6 to 0.95 for CD 34+ precounts that oscillated from 30 to 40 × 103CD34+ cells/ml, respectively. The Univariate Logistic Regression gave a probability of collecting ≥2.5 × 106CD34+ cells/kg that oscillated between 0.64÷0.98 for values of CD34+ cells processed from 6 × 106/kg to 8 × 106/kg, p <0.000. Sixty-three percent of patients reached the target dose of 2.5 × 106CD34+ cells/kg with only one LVP. Until now 12 patients have been transplanted and all have had a prompt and complete lasting recovery. These results confirm the efficacy of LVP in harvesting hemopoietic progenitors and their ability in reconstituting hemopoiesis of transplanted patients, enabling the estimation of CD34+ precounts and CD34+ cells processed values, highly predictive for the collection of ≥2.5 × 106CD34+ cells/kg. Furthermore, the Logistic Model suggests that the best strategy to plan a successful CD34+ cell collection procedure is to identify for each patient the amount of CD34+ cells/kg to be processed rather than the fixed processing of 3÷5 blood volumes in all patients.

The bisdioxopiperazine ICRF 187 is a potent intracellular chelating agent which effectively diminishes Adriamycin cardiotoxicity without compromising its antitumor activity. Our study aimed at verifying whether ICRF 187 can modulate the cytotoxic action of cisplatin (CDDP) on ovarian cancer cells. We used the A2780 ovarian cancer cell line and a subline resistant to CDDP (A2780-CDDP) obtained in our laboratory by continuous exposure of the parental cells to progressively increasing CDDP doses. In both cell lines ICRF 187 (0.1-0.5 μg/ml) used in combination with CDDP (0.01-1 μg/ml) produced a dose-dependent reduction of CDDP IC50(the concentration inhibiting 50% of cell growth). Moreover, when ICRF 187 was used in combination with CDDP, analysis of the data by the isobole method showed that the combination of the two drugs produced a synergistic antiproliferative activity in both cell lines, with a CDDP potentiation up to fivefold. Our in vitrodata show that ICRF 187 can synergize with CDDP. Prospective clinical trials are now needed to verify whether the addition of ICRF 187 to CDDP-containing regimens will result in an improved clinical response in ovarian cancer.

We have monitored the serum concentrations of hematopoietic growth factors (HGFs; ie, stem cell factor [SCF], leukemia inhibitory factor [LIF], interleukin-3 [IL-3], IL-6, IL-8, and granulocyte colony- stimulating factor [G-CSF]) in 15 lymphoma/leukemia and 6 ovarian cancer patients undergoing autologous bone marrow (BM) or peripheral blood (PB) stem cell transplantation (SCT). Thus, the analysis was performed during and after high-dose chemotherapy (from day -6 to day - 1), at the time of SCT (day 0), and thereafter (through day +17). Despite the heterogeneity of these patients and their conditioning regimens, a consistent kinetic pattern was observed for all analyzed cytokines. Particularly, (1) SCF serum concentration did not significantly fluctuate. (2) High levels of LIF (approximately 250 to 450 pg/mL) before chemotherapy rapidly declined to markedly lower concentrations (approximately 10 ng/mL) starting from day -1 through day +17; (3) conversely, IL-3 level was low before treatment, sharply increased during chemotherapy, and rapidly returned to base-line level after SCT. Hypothetically, the sharp LIF decrease and IL-3 increase during chemotherapy may underlie the induction of stem cell cycling and differentiation caused by hematopoietic ablation. Furthermore, (4) IL-6 concentration was low before and immediately after chemotherapy, but increased starting from day +5, peaked at day +6 through 9 and then declined to baseline level from day +10 onward; (5) a strictly similar pattern was consistently observed for both G-CSF and IL-8 levels, in agreement with our previous studies. It is relevant that peak IL-6, G- CSF, and IL-8 concentrations were directly correlated to peak neutrophil numbers in the recovery phase, thus suggesting an important role for these cytokines in granulocyte rescue; in line with this interpretation, hematologic patients undergoing PBSCT (10 of 15) exhibited higher peaks of IL-6, G-CSF, and IL-8 and a more pronounced increase of neutrophil/platelet number than did hematologic cases undergoing BMSCT (5 of 15). Altogether, these studies indicate a coordinate pattern of cytokine release during hematopoietic ablation/recovery after chemotherapy and autologous SCT, the fluctuations of LIF and IL-3 levels during chemotherapy are seemingly related to stem cell recruitment, whereas the post-SCT increase of IL- 6, G-CSF, and IL-8 may underlie the neutrophil recovery.

The present report describes the non-haematological toxicity and the influence of growth factor administration on haematological toxicity and haematopoietic recovery observed after high-dose carboplatin (1200 mg m(-2)), etoposide (900 mg m(-2)) and melphalan (100 mg m(-2)) (CEM) followed by peripheral blood progenitor cell transplantation (PBPCT) in 40 patients with high-risk cancer during their first-line treatment. PBPCs were collected during the previous outpatient induction chemotherapy programme by leukaphereses. CEM administration with PBPCT was associated with low non-haematological toxicity and the only significant toxicity consisted of a reversible grade III/IV increase in liver enzymes in 32% of the patients. Haematopoietic recovery was very fast in all patients and the administration of granulocyte colony-stimulating factor (G-CSF) plus erythropoietin (EPO) or granulocyte-macrophage colony-stimulating factor (GM-CSF) plus EPO after PBPCT significantly reduced haematological toxicity, abrogated antibiotic administration during neutropenia and significantly reduced hospital stay and patient's hospital charge compared with patients treated with PBPCT only. None of the patients died early of CEM plus PBPCT-related complications. Low non-haematological toxicity and accelerated haematopoietic recovery renders CEM with PBPC/growth factor support an acceptable therapeutic approach in an adjuvant or neoadjuvant setting.

It has been demonstrated that the flavonoid quercetin (3,3',4',5,7-pentahydroxyflavone; Q) inhibits the growth of several cancer cell lines. There is evidence suggesting that the antiproliferative activity of this substance is mediated by the so-called type II estrogen-binding, site (type II EBS). We looked for the presence of type II EBS and the effect of Q on the proliferation of an Adriamycinresistant estrogen-receptor-negative human breast-cancer cell line (MCF-7 ADRr). By whole-cell assay using estradiol labelled with 6,7-tritium ([3H]-E2) as a tracer, we demonstrated that MCF-7 ADRr cells contain type II EBSs. Competition analysis revealed that diethylstilbestrol (DES) and Q competed with similar potency for [3H]-Es binding to type II EBSs. The antiestrogen tamoxifen (TAM) competed for type II EBSs, albeit to a lesser extent than either DES or Q. Growth experiments demonstrated that Q and DES exerted a dose-dependent inhibition of cell proliferation in the range of concentrations between 10 nM and 10 µm, whereas TAM was less effective. Q could also inhibit colony formation in a clonogenic assay. Our results indicate that multidrug-resistant estrogen-receptor-negative MCF-7 cells express, type II EBSs and are sensitive to the inhibitory effect of Q. This substance could be the parent compound of a novel class of anticancer agents.

Hemopoietic CD34+ progenitors were isolated by immunomagnetic method from normal bone marrow (BM) or from peripheral blood (PB) of patients with non-Hodgkin's lymphoma treated with chemotherapy and granulocyte colony-stimulating factor (GCSF). Aliquots were seeded in longterm cultures (LTC) on bone marrow-derived stromal layers; non-adherent and adherent clonogenic content of the cultures was assayed weekly. The final recovery and the clonogenic efficiency of the CD34+ cells were sligthly higher in PB samples than in BM controls. In long term cultures PB cells sustained hemopoiesis as much as BM cells; at week 3 and 4 PB total mononuclear cells and CD34+ cells showed a non-adherent cell recovery higher than the respective BM controls. Furthermore, PB CD34+ cells were expanded in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF alone or combined with interleukin 3 (IL3), stem cell factor (SCF), interleukin 1 (IL 1), interleukin 6 (IL6). The combination of GM-CSF, IL3, SCF, IL 1 and IL6 produced the maximum increase of both mononuclear cells (30-fold) and granulocyte-macrophage colony forming units (CFU-GM) (4.6-fold) after 7 days of cultures; yet after 14 days a strong decrease of the CFU-GM occurred. These data suggest that G-CSF following chemotherapy mobilizes both early and committed hemopoietic progenitors.

Several authors have reported a faster immunological and hemopoietic post- transplant reconstitution using autologous peripheral blood stem cell (PBSC) than using autologous bone marrow stem cells. A large number of PBSC can be collected by leukapheresis during the hematological recovery after induction or salvage chemotherapy. In our experience we demonstrated that several separators, even if they have different results in mononuclear cell (MNC) yields, red blood cell and platelet contaminations, are able to collect PBSC for autotransplantation in patients with several malignant diseases and different status of disease. Eighty three patients were submitted to 590 leukapheresis procedures using 4 different blood cell separators: the results showed that all employed protocols are efficient in the collection of peripheral MNC even if after the widespread use of granulocytecolony stimulating factor (G-CSF) in the harvesting phase, the blood cell separator efficiency in terms of MNC is reduced. The use of GCSF in combination with other growth factors, during chemotherapy mobilization could simplify, in the future, this therapeutical program even if improvements in the efficiency of PBSC collection protocols are required.

A retrospective study was undertaken to assess the factors affecting the yield of peripheral blood stem cell (PBSC) collections after chemotherapy. Fifty-five patients with malignancies, observed in 4 Italian Institutions from January 1987 to June 1991 were eligible for evaluation. This series included 19 non-Hodgkin lymphoma, 11 multiple myeloma, 9 ovarian cancer, 7 Hodgkin disease, 7 acute non-lymphocytic leukemia, 1 acute lymphoblastic leukemia, 1 neuroblastoma. Five hundred and twenty two PBSC collections were performed on 55 patients after a median of 18 days after the start of chemotherapy. The yields of PBSC collections were related to the dose of cytoreductive chemotherapy exploited for PBSC mobilization and to the number of circulating white blood cells, colony forming unit granulocyte/macrophage (CFU-GM) and the percentage of monocytes at the time of collection.Forty-eight patients out of 55 transplanted (87%) had rapid, complete and sustained engraftment. Three patients (5%) died of transplant related complications.

We have analyzed the results of a multicenter study on peripheral blood stem cell transplantation (PBSCT) performed on 55 patients suffering from various neoplastic diseases. After myeloablative therapy, they received a mediam of 6.8x108/kg MNC and 11.4x104/kg CFU-GM harvested by a median of 9 apheresis after mobilization with chemotherapy alone. As of date, 34 of the 55 patients are alive and 28 of them are in continuous complete remission after a follow-up of 30 months. The probability of survival was related to the disease status at transplant, CR/PR vs. PD (p= 0.0001) and the bone marrow involvement, BM-vs. BM+ (P=0.009). Furthermore, a comparative study on speed of engraftment and clinical management was conducted on the 55 PBSCT patients as well as on 41 autoBMT and 52 alloBMT patients. Days to reach WBC> 1.0x109/L, PMN> 0.5x109/L and PLT> 50x109/L was 12/14/33 for PBSCT, 17/20/23 for ABMT and 15/16.5/18 for BMT, respectively. Days with fever >38° C, systemic antibiotic therapy and length of hospitalization was 3/12/36 for PBSCT, 5/18.5/42 for ABMT and 9/25/46 for BMT respectively.

We evaluated the protective ability of amifostine on peripheral blood mononuclear cell (PBMC)-derived colony-forming unit (CFU) and PB CD34+ cells which were previously exposed in vitro to etoposide, carboplatin, doxorubicin and taxotere. Amifostine pretreatment protected PBMC-derived CFU from the toxic effect of etoposide, carboplatin and taxotere. A significant detrimental effect was exerted by amifostine on the growth of doxorubicin-treated PBMC-derived CFU. Liquid cultures of PB CD34+ cells reproduced faithfully the effects observed on growth of PBMC-derived CFU and confirmed amifostine chemoprotection against etoposide and carboplatin with its detrimental effect on doxorubicin-treated progenitors. Combining the data of viable cell count, cytometric estimation of apoptosis, cell cycle and viable cell replication rate, we found that amifostine protects from etoposide and carboplatin toxicity mainly through a mechanism of cell rescue. Conversely, the detrimental effect of amifostine on the growth of doxorubicin-treated PB CD34+ cells is apparently due to an increased G2/M arrest. In conclusion, amifostine protects haematopoietic progenitors from etoposide, carboplatin and taxotere. Progenitor rescue is the mechanism through which amifostine reduced etoposide and carboplatin toxicity.

Recovery phase infection patterns in 55 patients who had undergone autologous blood progenitor cell transplantation (ABPCT) were evaluated retrospectively. The results were compared to those obtained in a group of 41 patients who received autologous bone marrow transplantation (ABMT). Fever related to documented or suspected infection developed in 38 of 55 patients in the ABPCT group and in 37 of 41 in the ABMT group (p<0.05). The percentages of patients with positive blood cultures did not differ significantly (ABPCT, 8/55 vs. ABMT, 8/41, p>0.05). However, fewer acquired systemic fungal infections (1/55 vs. 5/41, p<0.05) as well as fewer days of antibiotic usage were observed in the ABPCT group.

Blood donation allowed by cell separators can offer higher performance and higher yield to guarantee better quality and pureness of collected products. New systems for the collection of platelet concentrate (PC) and packed red blood cells (PRBC) are currently available. The aim of our work was to test the possibility of preparing PC routinely from normal apheresis donors in a minimum amount of time while providing a second product. Over a 3-month period we performed 40 procedures using the Hemonetics MCS3P blood cell separator and the Dideco Excel. The mean values of platelet yield were 2.8 x 1011(range 1.4-4.1) with the MCS3P and 3.49 x 1011(range 2.9-3.9) with the Excel, in a plasma volume of 240 ml and 215 ml respectively; the PRBC units were added with SAG-Mannitol allowing a storage time of 42 days. Collection times were 71’ and 48’ respectively. Donor tolerance was analogous to phateletapheresis or plasmapheresis.

Two consecutive trials were conducted to evaluate a VHDCT programme with autologous bone marrow (ABM) or perlpheral stem cell (APSC) support in previously untreated OVCA patients (Pts) with macroscopic (0.5–2cm) residual tumor. Fifthy-one pts (median age 44; stage IIIC 78%) underwent: Induction chemotherapy (Indct) (2–4 cycles of a cisplatin and cyclophosphamide combination +/- G-CSF) with ABM and/or APSC hsrvestlng (H) foflowed by intensification (Int) CT (one course of a platinum, etoposide combination +/- L-PAM, +/-G-CSF&EPO) In the absence of clinical progression. 2nd-look laparotomy was performed in complete responders. 42 (82%) and 39 (76%) pts are evaluable for toxicity and pathological response (PR), respectively while 6 pts are still on treatment, 3 progressed when on treatment, 2 are awaiting 2nd look. One toxic death occurred due to systemic mycosis in a pt undergoing ABM transplantation (T). The number of leukaphereses required for adequate APSCH decreased after G-CSF Incorporation. Duration of BM aplasia progressively decressed lor pts receiving ABMT. APSCT and APSCT + G-cSF & EPO, respectively. 90% pR (CR 44%, PR 41% of which 36% microscopic) was revealed at 2nd-1ook. A median follow up of 28 (2–79) months has been reached from diagnosis. Only 2 of the 19 pathologically complete responders (6 with a 5 year follow-up from 2nd-look) have so far relapsed without further therapy. Treatment proved to be feasible with acceptable toxicity when APSCT + G-CSF & EPO were used. The disease-free Interval would seem to be longer than expected in this pt subset. These data, if confirmed after a prolonged observation, warrants further investigation (in a randomized setting) on the possible therapeutic impact of this new approach on chemosensitive tumors.