Anna Gasperi-Campani

DNA-nuclease activity of the single-chain ribosome-inactivating proteins dianthin 30, saporin 6 and gelonin

The single‐chain ribosome‐inactivating proteins (sc‐RIPs) from plant origin are antiviral and antiproliferative agents employed in the preparation of immunotoxins. Similarly to the A‐chains of ricin, sc‐RIPs act as rRNA N‐glycosidases. We demonstrate here that dianthin 30, saporin 6 and gelonin exert a specific nuclease activity on supercoiled DNA. Four specific sites of cleavage introduced by dianthin 30 and by saporin 6 and two specific sites of cleavage introduced by gelonin have been identified and mapped in pBR322.

The nuclear localization signal is required for nuclear GPER translocation and function in breast Cancer-Associated Fibroblasts (CAFs).

Cancer associated fibroblasts (CAFs) actively contribute to the growth and invasion of cancer cells. In recent years, the G protein estrogen receptor (GPER) has been largely involved in the estrogenic signals in diverse types of normal and tumor cells. In CAFs, GPER was localized into the nucleus, however the molecular mechanisms which regulate its nuclear shuttle remain to be clarified. In the present study, we demonstrate that in breast CAFs GPER translocates into the nucleus through an importin-dependent mechanism. Moreover, we show that a nuclear localization signal is involved in the nuclear import of GPER, in the up-regulation of its target genes c-fos and CTGF and in the migration of CAFs induced by estrogens. Our data provide novel insights into the nuclear localization and function of GPER in CAFs toward a better understanding of the estrogen action elicited through these key players of the tumor microenvironment.

Inhibition of protein synthesis by seed‐extracts A screening study

Powerful inhibitors of protein synthesis by 80 S ribosomes have been found in several plants. Of these, ricin (from the seeds of Ricinus communis) and abrin (from the seeds of Abrus precatorius) are highly toxic to animals or cells in vitro (see for review [ I] ) whereas PAP (from the leaves of Phytolaccu americana) [2,3], cretin II (from the seeds of 0oton tiglium) and curcin II (from the seeds of Jatropha curcas) [4] have low or no toxicity. All these inhibitors are proteins, and seem to act catalytically through the same mechanism, i.e., by irreversibly inactivating in a still unknown way the 60 S ribosomal subunit, which becomes unable to react with elongation factor 2 [2,5-71. The present study was undertaken to ascertain whether similar inhibitors were present in seeds of other plants, initially selected by their taxonomic proximity or for their analogous toxic properties. It was observed that extracts from the seeds of several plants had an inhibitory effect on protein synthesis in a cell-free system (a lysate of rabbit reticulocytes). The same extracts had scarce or nil effect on protein synthesis by whole Ehrlich ascites cells, and were not toxic to mice at doses much higher than those sufficient to inhibit protein synthesis in vitro.

Caveolin-1 silencing arrests the proliferation of metastatic lung cancer cells through the inhibition of STAT3 signaling

Cav-1 is an essential structural constituent of caveolae implicated in mitogenic signaling, oncogenesis, angiogenesis, neurodegenerative diseases and senescence. Its role as a tumor suppressor gene or as a tumor promoter seems to strictly depend on cell type and tumor stage/grade. The high expression of Cav-1 in some tumors in vivo, amongst which lung adenocarcinoma, is associated with increased tumor aggressiveness, metastatic potential and suppression of apoptosis. In the present study we investigated the role of Cav-1 in metastatic lung cancer proliferation. Cell lines were from metastatic lesions of lung adenocarcinoma (RAL) and of small cell lung carcinoma (SCLC-R1), in which we found Cav-1 expressed at high levels. Results show that siRNA-mediated down-regulation of Cav-1 caused stable arrest of proliferation in both cell lines. A marked reduction of cyclin D1 and of CDK4 expression was evident in the cells transfected with Cav-1 siRNA and consequently of phospho-Rb on ser(795) and ser(780). Furthermore, a significant decrease of the expression of phosphorylated AKT and of its down-stream effectors phosphorylated ERK and STAT3 was evident. Together, these findings indicate that Cav-1 silencing induces an arrest of human metastatic lung proliferation in vitro by a new inhibitory pathway in lung cancer and provide new insights into the molecular mechanisms underlying the pro-survival and tumor-promoting functions of Cav-1.

In vitro activity of taxol and taxotere in comparison with doxorubicin and cisplatin on primary cell cultures of human breast cancers

SummaryThein vitro activities of taxol and taxotere in comparison with cisplatin and doxorubicin were assessed in 30 primary tumor cultures from human breast cancers. Both taxanes were much more potent than cisplatin and doxorubicin. Taxotere was 3.1; 296, and 9.6-fold more cytotoxic than taxol, cisplatin, and doxorubicin respectively. The cytotoxic activity observed in our experiments confirms the potential clinical relevance of the two taxanes in the management of breast cancer.

Effect of modeccin on rat liver ribosomes in vivo.

1. Rat liver microsomes isolated at 6 and 12 h of poisoning with 3 x LD50 (0.3 microgram/100 g body wt.) of modeccin, the toxin of Adenia digitata, have a decreased capacity of protein synthesis in vitro. 2. A similar decrease of protein synthesis is observed with polysomes at 6 h of poisoning. Experiments with recombined ribosomal subunits demonstrate that this is due to inactivation of the 60 S ribosomal subunit. 3. At 6 h of poisoning there is a marked vesiculation and degranulation of the hepatocyte rough endoplasmic reticulum, which is completely fragmented at 24 h of poisoning. Hepatocyte mitochondria are swollen at 6 h and shrunk at 24 h of poisoning. 4. It is concluded that modeccin penetrates inside hepatocytes in vivo, and damages ribosomes in the same manner as it does in vitro. However, mitochondrial damage indicates that ribosomes may not be the only target of modeccin in vivo.

Molecular and Biological Features of Two New Human Squamous and Adenocarcinoma of the Lung Cell Lines

Two human cancer cell lines were established from metastatic lesions of an adenocarcinoma (RAL) and a squamous cell (CAEP) carcinoma of the lung. The clinical histories of the patients from whom the cell lines were derived are reported. The lines were maintained in continuous culture with doubling times of 65 (RAL) and 50 (CAEP) hours. The RAL and CAEP cell lines, whose morphology and ultrastructural features are presented, showed extensively rearranged karyotypes with modal number of 85 (RAL) and 98 (CAEP). In particular, chromosome 2 pentasomy and several clonal markers were evident in the RAL cells, whereas a telomeric deletion of chromosome 1, del (1)(q32), was observed in the CAEP cells. The morphologic data were confirmed by high expression of specific antigens for each histotype. A marked positivity of the neuron-specific enolase (NSE) levels was evident by immunoenzymatic assays in the cell lines cytosol with respect to those present in the respective patients sera. No amplification or rearrangements were evident in the CMYC, LMYC, NMYC, INT-2, ERBB2, HRAS, KRAS, MOS, HST-1 genes by Southern blotting analysis in each cell line. Point mutations in exon 1 of KRAS and in exon 7 of TP53 were evident by polymerase chain reaction (PCR)-DNA sequencing in the RAL cell line, whereas no alterations were present in the HRAS and RB genes. The four genes studied did not show point mutations in the CAEP cell line. The RAL cell line was resistant to all the drugs tested, whereas the CAEP cells were sensitive to vinblastine. These cell lines may represent useful experimental models to investigate lung cancer biology and anticancer drug response.

Loss of heterozygosity at pseudoautosomal regions in human breast cancer and association with negative hormonal phenotype

To determine the possible involvement of X-linked genes in breast cancer, a group of human sporadic breast carcinomas were analyzed for loss of heterozygosity (LOH) at 12 polymorphic loci distributed along the whole chromosome X. LOH by at least one marker was observed in 14 of 46 informative cases and two regions of consistent LOH in 10 of 14 (71.4%) were identified at pseudoautosomal regions (PAR). Allelic losses in these regions significantly correlated with the absence of estrogen receptors (P<0.05) and concordant absence of either estrogen (ER) or progesterone (PgR) receptors (P<0.05). The clinicopathological parameters evaluated, (like age, menopausal status, histological type, tumor size, nodal status, grading, ploidy, labeling index, and S-phase fraction), were independent from the LOH present in the PAR regions of X chromosome. This study suggests a role as a prognostic factor for LOH and ER(-)/PgR(-) when associated and provides some additional support for the existence of candidate tumor suppressor gene on PAR regions, whose alteration may play a role in breast cancer development and progression.

Chromosomal Alterations, Biological Features and In Vitro Chemosensitivity of SCLC-R1, a New Cell Line from Human Metastatic Small Cell Lung Carcinoma

A new human cancer cell line was established from a metastatic lesion of a small cell lung carcinoma (SCLC-R1) and maintained in continuous culture with a doubling time of 62 h. The SCLC-R1 line, whose ultrastructural features are presented, showed a diploid DNA content, a translocation involving chromosome 16 [t(16;?)(q24;?)] and noticeable deletions in the FHIT (fragile histidine triad) region in the short arm of chromosome 3 [del(3)(p14)] and in the telomeric region of the short arm of chromosome 12 [del(12)(p13)]. The involvement of 12p in metastatic small cell lung cancer is reported here for the first time. No amplification or rearrangements were evident in the c-myc, L-myc, N-myc, int-2, c-erbB-2, H-ras, K-ras, c-mos, and hst-1 genes by Southern blot analysis. Wild-type p53, RB, K-ras and H-ras genes were evident by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. The neuron specific enolase (NSE) level was much higher in the cell lines cytosol than in the patients serum and the cell line also had high expression of chromogranin A and cytokeratin 19. SCLC-R1 cells were sensitive to cisplatin, carboplatin and doxorubicin. The clinical history of the patient from whom the cell line was derived is reported. The characteristics of this new cell line indicate it to be a useful experimental model to investigate lung cancer biology and anticancer drug response.

A new cell line from human infiltrating ductal carcinoma of the breast : establishment and characterization

We established a novel cancer cell line (MAST) from the ascitic fluid of a metastatic infiltrating ductal carcinoma of the breast. The epithelial and neoplastic nature of the MAST cells was confirmed by ultrastructural analysis. The cell line was maintained as a monolayer with a doubling time of about 68 h, and it possessed an abnormal karyotype with a modal chromosome number of 60, a trisomy of chromosome 18 and other unidentified rearranged chromosomes. Among the markers consistently found in MAST metaphases, we noted a t(14; 14) and a very large subtelocentric, a large satellited acrocentric and a very large submetacentric chromosome with striking fluorescent bands. Immunoenzymatic assay demonstrated that the MAST cell line was positive for estrogen and progesterone receptors. The in vitro drug-sensitivity assay showed a marked resistance of the cell line to 5-fluorouracil and 4-hydroperoxycyclophosphamide and a moderate resistance to etoposide and 4′-epidoxorubicin. The molecular analysis showed a four- to sixfold amplification of the c-myc gene and no amplification or rearrangement of theint-2, c-erbB-2, c-Ha-ras, c-mos andhst-1 genes.