Giuliano Mazzini

Expression of p53 protein during the cell cycle measured by flow cytometry in human leukemia

The nuclear protein p53 has been reported to be associated with cell transformation and/or proliferation so that the study of p53 expression in human malignancy has potentially important clinical implications. We have analyzed the p53 expression in mitogen-stimulated and nonstimulated human lymphocytes, in several human leukemia cell lines (Molt-4, Raji, Daudi, HL-60, KG-1, K562 and U937) and in fresh bone marrow (BM) cells. Simultaneous differential staining of p53 (identified by a FITC-labeled monoclonal antibody) versus DNA (stained with propidium iodide, PI), followed by bivariate analysis with flow cytometry (FCM) made it possible to evaluate p53 expression with respect to cell position during the cell cycle. The data show that in stimulated lymphocytes p53 is progressively accumulated during the G1, S and G2-phases, while in non-stimulated conditions most cells are remaining in G0/G1 and express p53 to a lesser degree. This suggests that expression of p53 is more correlated with cell growth than with entrance into (or progression through particular phases of) the cell cycle. Cells from acute lymphoblastic leukemia (ALL) and Burkitts lymphoma cell lines express elevated levels of p53, while all examined human acute myeloid leukemia cell lines synthesize negligible p53 protein. Understanding the variations in p53 expression in different types of human leukemia may provide some insight into the biologic roles of p53 in normal and malignant cells.

Modified expression of Bcl-2 and SOD1 proteins in lymphocytes from sporadic ALS patients

Markers of oxidative stress have been found in spinal cord, cortex, cerebrospinal fluid, and plasma of SALS patients. Mitochondrial and calcium metabolism dysfunction were also found in peripheral lymphocytes from SALS patients. In this study, we demonstrate that lymphocytes from SALS patients are more prone to undergo alteration of cell membrane integrity both in basal conditions and following oxidative stress induced by H2O2 treatment. The expression of the antioxidant proteins, Bcl-2, SOD1 and catalase in basal conditions, was significantly lower in lymphocytes from SALS patients than in lymphocytes from age and sex matched controls. Exposure to H2O2 induced a time-dependent decrease of Bcl-2 and SOD1 in control lymphocytes. Conversely, the levels of these proteins remained unchanged in SALS lymphocytes even after 18 h stress. Catalase expression was not significantly modified by oxidative stress. Our results demonstrate that two factors involved in the genesis and/or progression of the familial form of the disease with SOD1 mutation are altered also in the sporadic form of ALS and suggest that the oxidative stress protection pathway is deregulated in lymphocytes from ALS patients.

Conformationally altered p53 : a novel Alzheimer's disease marker?

The identification of biological markers of Alzheimers disease (AD) can be extremely useful to improve diagnostic accuracy and/or to monitor the efficacy of putative therapies. In this regard, peripheral cells may be of great importance, because of their easy accessibility. After subjects were grouped according to diagnosis, the expression of conformationally mutant p53 in blood cells was compared by immunoprecipitation or by a cytofluorimetric assay. In total, 104 patients with AD, 92 age-matched controls, 15 patients with Parkinsons disease and 9 with other types of dementia were analyzed. Two independent methods to evaluate the differential expression of a conformational mutant p53 were developed. Mononuclear cells were analyzed by immunoprecipitation or by flow-cytometric analysis, following incubation with a conformation-specific p53 antibody, which discriminates unfolded p53 tertiary structure. Mononuclear cells from AD patients express a higher amount of mutant-like p53 compared to non-AD subjects, thus supporting the study of conformational mutant p53 as a new putative marker to discriminate AD from non-AD patients. We also observed a strong positive correlation between the expression of p53 and the age of patients. The expression of p53 was independent from the length of illness and from the Mini Mental State Examination value.

Karyotype analysis of the euploid cell population of a mouse embryonic stem cell line revealed a high incidence of chromosome abnormalities that varied during culture.

It is common knowledge that mouse embryonic stem cell (mESC) lines accumulate chromosomal changes during culture. Despite the wide use of mESCs as a model of early mammalian development and cell differentiation, there is a lack of systematic studies aimed at characterizing their karyological changes during culture. We cultured an mESC line, derived in our laboratory, for a period of 3 months investigating its chromosome complement at different times. About 60% of the metaphases analysed were euploid throughout the culture period but, from passage 13, only 50% of the euploid metaphases had a proper chromosome complement. The remaining 50% showed chromosome abnormalities, mainly gain or loss of entire chromosomes, both within the same passage and among different passages analysed. The very heterogeneous spectrum of abnormalities indicates a high frequency of chromosome mutations that arise continuously during culture. The heterogeneity of the aberrant chromosome constitution of 2n = 40 metaphases, observed at different passages of culture, might be due either to their elimination or to a shift towards the hypoeu- or hypereuploid population of those metaphases that accumulate further chromosome abnormalities. The stability of the frequency of eu-, hypoeu- and hypereuploid populations during culture might, however, be due to the elimination of those cells that carry a high mutational burden. Based on our results, we suggest that karyotype analysis of the euploid cell population of mESC lines is necessary when such lines are used in the production of chimeric mice, for their contribution to the germ line, or when they are differentiated into specific cell types.

Cell kinetics of human brain tumors: in vivo study with bromodeoxyuridine and flow cytometry

Bromodeoxyuridine (BUDR) is a thymidine analog which is incorporated into the DNA of proliferating cells. Since the dose of BUDR needed to label cells is not toxic, cell labelling can be accomplished in vivo, by infusing the substance in patients. A monoclonal antibody against BUDR is then used to identify BUDR-labelled cells. The same cell population can also be stained for DNA content with propidium iodide (PI). Using bivariate flow cytometry (FCM) for measurements, both the percentage of BUDR-labelled cells and their total DNA content can be evaluated. This technique allows one to obtain the labelling index (LI) and the DNA synthesis time (TS). The potential doubling time (Tpot) and the fractional turnover rate (FTR) can be mathematically derived, so that a complete picture of tumor growth can be obtained. Our aim was to ascertain whether this method is clinically applicable and whether the kinetic values obtained are reliable. We studied 22 patients with benign and malignant brain tumors, and observed no immediate toxicity from BUDR administration. The BUDRLI obtained ranged from 0.9% to 3.9% (median: 2.0%) in meningiomas and from 3.8% to 7.6% (median: 6.3%) in malignant gliomas (P less than 0.01). The fraction of S-phase cells determined with the BUDR FCM technique was statistically similar to that found by single DNA flow cytometric analysis performed on duplicate samples of both benign and malignant brain tumors. The TS obtained in malignant gliomas ranged from 10.5 to 227 h (median: 12.8). The calculated Tpot ranged from 7.6 to 26.8 days (median: 11.6), and the calculated FTR ranged from 3.7 to 13.1 cells/100 cells/day (median: 8.8). These data suggest that in vivo BUDR infusion coupled with FCM can be performed in clinical settings, and it is reliable and can easily be used for kinetic studies in clinical trials aimed at evaluating the prognostic relevance of proliferative parameters and in planning tumor treatment.

Proliferative activity of bone marrow cells in primary dysmyelopoietic (preleukemic) syndromes

The proliferative activity of bone marrow cells was studied in 24 patients with primary dysmyelopoiesis by means of flow cytometry and 3H‐IdR autoradiography. Abnormal DNA content was found in two cases with aneuploid karyotypes. DNA content typical of a diploid population was observed in all patients with normal karyotype and in three patients with chromosomal aberrations. The fraction of bone marrow cells in S‐and G2‐phase was higher in primary acquired sideroblastic anemia and refractory anemia (without excess of blasts) than in refractory anemia with excess of blasts and chronic myelomonocytic leukemia. Regardless to the diagnosis, the patients with low fraction of cells in S‐ and G2‐phase had short survival time and showed high rate of evolution into acute nonlymphoblastic leukemia. The labeling (LI) and mitotic (MI) indexes of both erythroblasts and granulocytic cells were decreased in nearly all patients. The lowest values of LI and MI of the granulocytic compartment were found in the patients who subsequently developed acute leukemia. These data suggest that cytokinetic analysis allows investigators to achieve useful information on the stage of disease in the dysmyelopoietic syndromes.

DNA ligase I deficiency leads to replication-dependent DNA damage and impacts cell morphology without blocking cell cycle progression.

ABSTRACT 46BR.1G1 cells derive from a patient with a genetic syndrome characterized by drastically reduced replicative DNA ligase I (LigI) activity and delayed joining of Okazaki fragments. Here we show that the replication defect in 46BR.1G1 cells results in the accumulation of both single-stranded and double-stranded DNA breaks. This is accompanied by phosphorylation of the H2AX histone variant and the formation of γH2AX foci that mark damaged DNA. Single-cell analysis demonstrates that the number of γH2AX foci in LigI-defective cells fluctuates during the cell cycle: they form in S phase, persist in mitosis, and eventually diminish in G1 phase. Notably, replication-dependent DNA damage in 46BR.1G1 cells only moderately delays cell cycle progression and does not activate the S-phase-specific ATR/Chk1 checkpoint pathway that also monitors the execution of mitosis. In contrast, the ATM/Chk2 pathway is activated. The phenotype of 46BR.1G1 cells is efficiently corrected by the wild-type LigI but is worsened by a LigI mutant that mimics the hyperphosphorylated enzyme in M phase. Notably, the expression of the phosphomimetic mutant drastically affects cell morphology and the organization of the cytoskeleton, unveiling an unexpected link between endogenous DNA damage and the structural organization of the cell.

Isolation of rare circulating tumor cells in cancer patients: technical aspects and clinical implications

Circulating tumor cells (CTCs) may be detected in the blood of patients with epithelial tumors using different analytical approaches. The relative number of CTCs is low and they include a heterogeneous population of cells with diverse biological and molecular characteristics, often different from those of the respective primary tumor. Until recently, they have been difficult to detect and, even though discordant results have been reported when different methods of detection were used, they may provide prognostic and predictive information. Several antibody- or molecular-based CTC detection methods have been developed, offering hope for individualized risk assessment by utilizing CTCs as biomarkers of disease progression and drug response. Pilot studies have also shown that by utilizing methods that permit, besides enumeration, a molecular characterization of CTCs, one could better identify high-risk patients, predict response to targeted therapies, analyze gene expression profiles (in order to identify new potential drug targets) and increase our knowledge of the metastatic process. In this article we review the techniques currently utilized for isolation and characterization of CTCs and we discuss their potential utility in clinical oncology focusing on the future perspectives in this field.

Cell cycle-related proteins: a flow cytofluorometric study in human tumors

We used 2‐parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor Trf‐r), the “cyclin” (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki‐67. FITC‐labeled antibodies against Trf‐r, PCNA, and the Ki‐67‐reactive antigen, as well as propidium iodide‐DNA distribution, were simultaneously measured on human leukemia HL‐60 and K562, and breast carcinoma MCF‐7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf‐r and PCNA and the 95% acetone fixation for Ki‐67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high‐quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf‐r is expressed mainly at the transition from G0/G1 to S‐phase. PCNA expression is prominent in late G1 and through S‐phase and decreases in G2‐M. The Ki‐67‐reactive antigen is widely distributed in G1, S, and G2‐M phases. Knowledge regarding the relationships between proliferation‐associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.

Electromagnetic enhancement of a culture of human SAOS-2 osteoblasts seeded onto titanium fiber-mesh scaffolds

The surface properties of a biomaterial are fundamental to determine the response of the host tissue. In the present study, we have followed a particular biomimetic strategy where electromagnetically stimulated SAOS-2 human osteoblasts proliferated and built a calcified extracellular matrix on a titanium fiber-mesh surface. In comparison with control conditions, the electromagnetic stimulation (magnetic field intensity, 2 mT; frequency, 75 Hz) caused higher cell proliferation and increased surface coating with type-I collagen, decorin, and osteopontin (9.8-fold, 11.3-fold, and 9.5-fold, respectively). Reverse transcriptase-polymerase analysis revealed the electromagnetically upregulated transcription specific for the foregoing matrix proteins and for the growth factor TGF-beta1. The immunofluorescence of type-I collagen, decorin, and osteopontin showed their colocalization in the cell-rich areas. The use of an electromagnetic bioreactor aimed at obtaining the surface modification of the biocompatible metallic scaffold in terms of cell colonization and coating with calcified extracellular matrix. The superficially modified biomaterial could be used, in clinical applications, as an implant for bone repair.