Laura Ottaggio

STROMAL DAMAGE AS CONSEQUENCE OF HIGH-DOSE CHEMO/RADIOTHERAPY IN BONE MARROW TRANSPLANT RECIPIENTS

Bone marrow transplant (BMT) relies on the engraftment of donor hemopoietic precursors in the host marrow space. Colony forming units-fibroblasts (CFU-f), the precursor compartment for the osteogenic lineage, are essential to hemopoietic stem cell survival, proliferation and differentiation. We have studied CFU-f in donors (aged 5 months to 62 years) and in patients who had received allogeneic BMT (aged 2 months to 63 years). In donor marrows we found an inverse correlation between CFU-f frequency and age. In BMT recipients CFU-f frequencies were reduced by 60%-90% (p < 0.05) and the numbers did not recover up to 12 years after transplant. Stromal reconstitution to normal levels was found only in patients < 5 years old. In all patients studied CFU-f post-BMT were of host origin. Patients with low CFU-f levels displayed also a decreased bone mineral density (p < 0.05) and significantly reduced levels of long-term culture-initiating cells (LTC-IC) (p < 0.05). Our study demonstrates that the marrow stromal microenvironment is seriously and irreversibly damaged after BMT. Donor cells do not contribute to reconstitute the marrow microenvironment, whose residual CFU-fs remain of host origin.

The presence of amplified regions affects the stability of chromosomes in drug-resistant Chinese hamster cells.

The stability of chromosomes carrying amplified CAD (carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) or DHFR (dihydrofolate reductase) genes was studied in V79 Chinese hamster cell derivatives resistant to PALA (N-phosphonacetyl-L-aspartate) and MTX (methotrexate), respectively. Cells were maintained in the presence of the selective drugs during the study. In both metaphase chromosomes and interphase nuclei, amplified regions were localized by in situ hybridization. In MTX-resistant cells, the amplification-bearing chromosome moved sluggishly at anaphase and gave rise to bud-shaped formations in interphase nuclei. It is suggested that these buds could eventually separate as micronuclei. In both MTX- and PALA-resistant cells, amplified DNA was observed in micronuclei in interphase and in displaced chromosomes in metaphase. Finally, amplification-bearing dicentric chromosomes were found in both drug-resistant cell lines. Cumulatively, these observations indicate that the presence of the amplified region in a chromosome renders it unstable: chromosomes bearing an amplified region tended to be excluded from cells, and rearrangements were more frequent than in normal chromosomes.

In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes

BackgroundTaxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species) were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established.ResultsCalli were successfully induced from stems, leaves and seeds grown in various hormone concentrations and combinations. The most suitable callus to establish suspension cell cultures was obtained from seeds. Media recovered from suspension cell cultures contained taxanes, and showed antiproliferative activity on human tumour cells. Taxol, 10-deacetyltaxol and 10-deacetylbaccatin III were the main taxanes identified. The level of Taxol recovered from the media of hazel cultures was similar to that found in yew cultures. Moreover, the production of taxanes in hazel cell cultures increased when elicitors were used.ConclusionHere we show that hazel cell cultures produce Taxol and taxanes under controlled conditions. This result suggests that hazel possesses the enzymes for Taxol production, which until now was considered to be a pathway particular to Taxus genus. The main benefit of producing taxanes through hazel cell cultures is that hazel is widely available, grows at a much faster rate in vivo, and is easier to cultivate in vitro than yew. In addition, the production of callus directly from hazel seeds shortens the culture time and minimizes the probability of contamination. Therefore, hazel could become a commercial source of Taxol and taxanes, both to be used as new therapeutic agents or as new precursors for Taxol semi-synthesis.

Taxanes from shells and leaves of Corylus avellana

Paclitaxel is an effective antineoplastic agent originally extracted in low yield from the bark of Taxus brevifolia. Although it was generally considered a particular metabolite of Taxus sp., paclitaxel was recently found in hazel cell cultures. The aim of the present work was to verify whether hazel differentiated tissues could be used as a commercial source of paclitaxel and other taxanes. Thus, shells and leaves of hazel plants were analyzed by ELISA and HPLC-MS. Both shell and leaf extracts contained taxanes. Among these, paclitaxel, 10-deacetylbaccatin III, baccatin III, paclitaxel C, and 7-epipaclitaxel were identified and quantified. Hazel extracts also showed biological activity, inhibiting metaphase to anaphase transition in a human tumor cell line. The level of total taxanes in leaves was higher than in shells collected in the same period from the same plants. However, the finding of these compounds in shells, which are considered discarded material and are mass produced by many food industries, is of interest for the future availability of paclitaxel and other antineoplastic compounds.

The yeast p53 functional assay: a new tool for molecular epidemiology. Hopes and facts.

The assumption of molecular epidemiology that carcinogens leave fingerprints has suggested that analysis of the frequency, type, and site of mutations in genes frequently altered in carcinogenesis may provide clues to the identification of the factors contributing to carcinogenesis. In this mini-review, we revise the development, and validation of the yeast-based p53 functional assay as a new tool for molecular epidemiology. We show that this assay has some very interesting virtues but also has some drawbacks. The yeast functional assay can be used to determine highly specific mutation fingerprints in the human p53 cDNA sequence. Discrimination is possible when comparing mutation spectra induced by sufficiently different mutagens. However, we also reported that the same carcinogen may induce distinguishable mutation spectra due to known influencing factors.

PRIMA-1 synergizes with adriamycin to induce cell death in non-small cell lung cancer cells.

p53‐dependent apoptosis is important for the efficacy of cancer treatment, and tumors carrying mutant p53 are often resistant to chemotherapy. Non‐small cell lung cancer (NSCLC) cells generally exhibit resistance to apoptosis following treatment with many cytotoxic drugs. The new molecule PRIMA‐1 appears to kill human tumor cells by restoring the transcriptional activity to mutated p53. We investigated the induction of apoptosis in response to this drug in three NSCLC cell lines carrying different p53 proteins: A549 (p53wt), LX1 (p53R273H), and SKMes1 (p53R280K). PRIMA‐1 alone did not trigger apoptosis but significantly reduced cell viability. However, in combination with adriamycin, PRIMA‐1 strengthen the adriamycin‐induced apoptosis in A549 and LX1. Interestingly, even in SKMes1 cells, the combined treatment triggered a strong PARP cleavage without DNA fragmentation. Our data suggest that in NSCLC cells, PRIMA‐1 may induce cell death through pathways other than apoptosis but may synergize with adriamycin to trigger an apoptotic response. J. Cell. Biochem. 104: 2363–2373, 2008.

Study on aneuploidy and p53 mutations in astrocytonias

To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed.

Alkaline DNA fragmentation, DNA disentanglement evaluated viscosimetrically and sister chromatid exchanges, after treatment in vivo with nitrofurantoin

Nitrofurantoin was not positive as a carcinogen in long term assays. In vitro it was positive in some short term tests and negative in others. We have examined Nitrofurantoin for its capability of inducing DNA damage in vivo. With the alkaline elution technique, Nitrofurantoin appeared clearly positive in all the tissues examined (liver, kidney, lung, spleen and bone marrow). In the liver we also observed some cross-linking effect. In bone marrow cells Nitrofurantoin was also clearly positive in terms of sister chromatid exchanges (SCEs) induction. DNA damage in vivo was also examined with a viscosimetric method, more sensitive than alkaline elution. With this method the results were essentially negative, suggesting that the two methods detect different types of damage. In view of its positivity in many organs and in two short term tests in vivo, the carcinogenic potential of Nitrofurantoin should be reconsidered.

Exposure of human lymphocytes and lymphoblastoid cells to simulated microgravity strongly affects energy metabolism and DNA repair

Exposure of freshly drawn lymphocytes and lymphoblastoid cells (LB and COR3) to simulated microgravity decreased the intracellular ATP concentration to 50%–40% of the value found in normal growth conditions. The decrease was reversible although recovery to normal values occurred only slowly both in lymphocytes and in lymphoblastoid cells. Poly(ADP‐ribose) polymerase (PARP ) activity was increased indicating that cells exposed to conditions of reduced gravitation experience stress. Exposure to microgravity forces cells into a condition of metabolic quiescence in which they appear to be particularly sensitive to subsequent exposures to a genotoxic agent. Thus, treatment of cells with the strong redox agent potassium bromate under microgravity conditions, indicated an impairment in repair of DNA 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), an oxidized derivative of deoxyguanosine. We conclude that gravitational modulation of the kind routinely obtained under laboratory conditions and during spaceflights is a stressful process to which cells appear to be extremely sensitive. These effects may reflect the physiological alterations observed in astronauts and in animals following spaceflights or exposure to conditions of simulated microgravity.

Chronic lymphocytic leukemia nurse-like cells express hepatocyte growth factor receptor (c-MET) and indoleamine 2,3-dioxygenase and display features of immunosuppressive type 2 skewed macrophages

Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3TYR705 phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients’ monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET+ and indoleamine 2,3-dioxygenase+ cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4+CD25high+/FOXP3+ T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of survival of the leukemic clone and indirectly by favoring T-cell immunosuppression.