Exosomes: novel effectors of human platelet lysate activity.

Despite the popularity of platelet-rich plasma (PRP) and platelet lysate (PL) in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF) released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC) treated with three different exosome concentrations (0.6 µg, 5 µg and 50 µg) showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and transforming growth factor beta 1 (TGF-ß1) as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies.

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 patient's 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.

Molecular genetics and in vitro sensitivity of a new human cell line, KKP, from a gastric adenocarcinoma.

A new cancer cell line (KKP) was established from an ascitic effusion of an advanced gastric adenocarcinoma, intestinal type. The line has been maintained in continuous monolayer culture with a doubling time of 48 hours for more than 2 years. KKP cells, whose ultrastructural features are presented, showed an aneuploid DNA content, a modal number of 53 chromosomes, and the presence of one double minute chromosome. The karyotype showed trisomies of chromosomes 7, 12, 13, and 14, tetrasomy of chromosome 18, a reciprocal translocation [t(1;20)(q21;p11.2)], and a [t(4;?)] rearrangement. No amplification or rearrangements were evident in the c-MYC, c-ERB B2, H-RAS, INT-2, HST-1, c-MOS, and K-RAS genes, whereas somatic rearrangements were present in the sequences corresponding to c-MET and cyclin E genes by Southern blotting analysis. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of P53 and RB genes did not reveal alterations or point mutations in the SSCP pattern of conformers. The chemosensitivity pattern assay of the KKP cell line indicated that it was sensitive to cisplatin, etoposide, and doxorubicin and resistant to 4'-hydroperoxycyclophosphamide. The clinical history of the patient from whom the cell line was derived is reported and compared with the results observed in the cell line in vitro. High levels of the tumor-associated antigens CEA (carcinoembryonic antigen) and CA19-9 were evident in the KKP cytosol, whereas the KKP spent culture medium maintained the same low levels of CEA and CA 19-9 found in the patient's serum. This new cell line may represent a useful tool for studying the biology of gastric cancer and for planning new therapeutic approaches.

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 line's cytosol than in the patient's 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.

Establishment and characterization of two new cell lines derived from human metastatic breast carcinomas.

Two human cancer cell lines (MA 2 and MA 3) were established from pleural effusions of infiltrating ductal carcinomas of the breast. The lines were maintained in continuous monolayer culture with doubling times of 70 (MA 2) and 78 (MA 3) hr for more than two years and possessed extensively rearranged abnormal karyo-types with modal chromosome number of 83 (MA 2) and 81 (MA 3) and DNA index values of 1.65 and 1.77, respectively. No amplifications or rearrangements were evident in the c-myc, int-2, c-erb B2, c-Ha-ras, or hst 1 genes in MA 2 and MA 3 cell lines. The clinical histories of the patients from whom the cell lines were derived are reported and compared with the results observed in the cell lines in vitro. The presence of CEA, CA 15-3, and MCA tumor markers observed in the primary tumor tissues was retained by the established cell lines. While the primary tumor tissues were ER+/PgR borderline+ (MA 2) and ER-/PgR+ (MA 3), the MA 2 line was ER+/PgR- and the MA 3 line remained ER-/PgR+. The MDR P-glycoprotein was not expressed either in primary tumor tissues or in the respective cell lines. High expression of cytokeratins 7, 18, and 19 was evident by immunohistochemical analysis in each cell line. whereas cytokeratins 8 and 17 were poorly or not at all expressed. The treatment history of the patients from whom the cell lines were derived involved CMF followed six months later by novantrone and cisplatin plus VP 16 (MA 2) and FEC followed four years later by CMF (MA 3). The chemosensitivity pattern assay of the cell lines indicated that the MA 2 line was sensitive to doxorubicin, cisplatin, and vinblastine, whereas the MA 3 line was sensitive to doxorubicin and cisplatin. The characteristics of these cell lines indicate them to be a good experimental model to investigate breast cancer biology and anticancer drug response.

Saporin 6 and lonidamine in primary cell cultures from human breast carcinomas: a synergistic effect.

Lonidamine (LND) is a potential chemotherapeutic agent which can positively modulate the efficacy of several antineoplastic agents. The ribosome-inactivating protein Saporin 6 (SO 6), which acts as a rRNA N-glycosidase and a DNA nuclease, has recently attracted interest as a novel potential anticancer and antiviral agent. Synergism between LND and SO 6 was previously demonstrated by us in the human metastatic MAST breast cancer cell line in vitro. In the present study, the antiproliferative effect of the drugs, either alone or in combination, was investigated in vitro at various concentrations in 17 primary cell cultures established from patients carrying infiltrating ductal carcinomas of the breast. Results indicate a strong synergistic effect in 11/17 cases, when LND was administered as a second drug. This is in agreement with previous results in the MAST cell line. Synergism was evident at SO 6 concentrations between 3.3 x 10(-10) and 1.7 x 10(-9) M.

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

The single-chain ribosome-inactivating proteins (scRIPs) 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.

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 the int-2, c-erbB-2, c-Ha-ras, c-mos and hst-1 genes.

In vitro potentiation by lonidamine of the sc-RIP saporin 6 effect in a human metastatic breast cancer cell line.

The ability of Lonidamine (LND), an energolytic derivative of indazole-carboxylic acid, to modulate the antiproliferative effect of the single-chain ribosome-inactivating protein Saporin 6 (SO 6) was investigated in the human MAST breast cancer cell line, recently established from an ascitic effusion of a ductal carcinoma, by analysis of protein synthesis inhibition and of colony formation in vitro. Different schedules were tested varying with regard to time of exposure (0-24 h), concentration of the drugs (0.01- > 10 micrograms/ml SO 6; 25-100 micrograms/ml LND) and sequence of administration (LND- > SO 6; SO 6- > LND; SO 6+LND). Results indicate that the marginal activity exerted here by each drug when tested independently is highly potentiated by the combination treatments, the cytotoxicity becoming significantly greater than that expected from an additive effect between the two drugs. In particular, a strong synergistic effect is obtained when SO 6 preceedes LND, with a reduction of the SO 6 IC 50 from 1.3 x 10(-7) M to 2.6 x 10(-9) M.

Diverse activity of sc-RIP saporin 6 on primary and metastatic melanoma cells in vitro.

The inhibitory effect of saporin 6, a single-chain ribosome-inactivating protein (sc-RIP) purified from the seeds of Saponaria officinalis, on the proliferation of human primary (MeWo, WM 164, SK MEL 28, MEM), cloned (MEM A9, A12, A13) and metastatic (M14) melanoma cells has been tested by protein synthesis and colony formation assays in vitro. Results indicate a marked difference in the sensitivity of primary and metastatic cells to the action of saporin 6, the latter being significantly more affected, both in treated and in pretreated cultures, with a high and specific response evident after 24 h of treatment and progressively increasing up to 72 h of culture with the drug (IC50 = 0.82 microgram/ml). This effect, which was dose-dependent in exponentially growing cells, was partially reversed upon removal of the inhibitor from the culture medium. No inhibitory effect was evident in the MeWo primary cells at the highest saporin 6 concentration used: the p170 glycoprotein-mediated mechanism is not involved in such a resistance pathway.

Inhibition of growth of breast cancer cells in vitro by the ribosome-inactivating protein saporin 6.

The antiproliferative activity of Saporin 6, a Ribosome-inactivating protein purified from the seeds of Saponaria officinalis has been tested on human breast cancer cells in vitro by the analysis (a) of colony formation in cells from surgical specimens from 27 patients bearing primary breast cancer and (b) of protein synthesis inhibition in the MCF/7 cell line. Results indicate a very high sensitivity of breast cancer cells from most patients to a short-term treatment with Saporin 6 at concentrations (10(-9) M), until now found effective only in acellular systems or after conjugation with monoclonal antibodies. On the contrary, the treatment of the human cell line MCF/7 indicate a very reduced sensitivity compared to fresh human neoplastic cells, with the necessity of a long lag for the effect to begin.

Effect of tulipin on cell cycle progression analyzed by BrdUrd incorporation.

The effect of tulipin, a protein from plant origin recently purified, on cell cycle progression has been analyzed in the sensitive EUE cells by BrdUrd incorporation. The cytofluorimetric results demonstrate that tulipin specifically interacts with the S phase, with a dose-dependent decrease of the total S phase cells and an increase of the G1/G2 cells after 4 h of treatment in the synchronized EUE cells, whereas in the asynchronous population it mainly causes a dose-dependent decrease in the incorporation of BrdUrd per cell.

Differential activity of saporin 6 on normal and leukemic hemopoietic cells.

The antiproliferative effect of saporin 6 (SO6), a ribosome-inactivating protein (RIP) purified from the seeds of Saponaria officinalis has been tested on three leukemic cell lines (K562, U937, and HL60), human normal bone marrow, and peripheral blood hemopoietic progenitor cells from normal subjects. In leukemic cell lines, SO6 appeared much more effective against erythrocytic than against monocytic and promyelocytic leukemic cells, as shown by protein synthesis assays carried out after up to 72 h of culture. Among the normal hemopoietic progenitor cells, erythroid burst-forming units were the most affected, with results similar to those observed in the erythroid leukemic cell line, both in treated and in pretreated cultures, with strong damage after 24 h of exposure to SO6. On the other hand, granulocyte-macrophage colony-forming units (CFU-GM) from bone marrow were significantly more affected than the myeloid leukemic cell lines after permanent treatment with the inhibitor, the damage being significantly lower after an exposure of 24 h. CFU-GM from peripheral blood and megakaryocyte CFU showed an intermediate sensitivity after 24 h of exposure to SO6, similar to that of the other normal precursors after permanent treatment with the drug.

Mapping of the Emery-Dreifuss gene through reconstruction of crossover points in two Italian pedigrees.

Two unrelated pedigrees, which show recurrence of Emery-Dreifuss muscular dystrophy (EDMD) in three generations, have been studied using 13 X-linked DNA polymorphisms and somatic cell hybrids to establish the phase of the corresponding alleles in some obligate carriers. The reconstruction of cross-over points on the X chromosomes carrying the EDMD gene excludes from mapping most regions of the X chromosome except for the terminal portion of Xq. Pooled linkage data from the two pedigrees confirm the linkage previously reported with locus DXS15. A cross-over in a carrier female suggests that the EDMD gene is probably located distally to DXS15. In addition the recombinant meioses from one of the two pedigrees suggest the following order for some Xq polymorphic loci: DXS1 (DXYS1-DXS178) DXS42 (F9-DXS15).

A second genetic locus for autosomal dominant polycystic kidney disease.

Hitherto, mutations that lead to autosomal dominant adult-type polycystic kidney disease have been found to be linked to the alpha-globin genes on the short arm of chromosome 16. In an Italian family, absence of linkage between the disease mutation and alpha-globin indicates that the condition can be caused by mutations in a second gene. The clinical features of the disease in this Italian family are indistinguishable from those found in the "linked" families. The finding that there are two polycystic kidney disease genes means that linkage must be demonstrated independently in each family before predictive tests with DNA probes can be used reliably.

Italian experience regarding the prevention of Duchenne and Becker muscular dystrophies.

The indirect approach to carrier detection and prenatal diagnosis of Duchenne and Becker muscular dystrophies based on the study of DNA polymorphisms closely linked to this gene has been followed by five Italian laboratories in the study of 106 pedigrees. Out of 354 women studied up to 1 May 1987, 147 were identified as carriers because of pedigree information and/or of increased creatine phosphokinase (CPK) values. Of the remaining 207, 184 could be assigned to three arbitrarily defined risk categories (low, intermediate and high) using linkage analysis. This disaggregation of women at risk is clearly more useful than that defined before DNA analysis, in which the same 184 women could be assigned only to the low or intermediate risk categories. Prenatal diagnosis was theoretically possible in 90% of carrier women, and was actually performed in 14 pregnancies, which led to the identification of four affected male foetuses, one also having Down syndrome.

Hereditary thrombophilia: identification of nonsense and missense mutations in the protein C gene.

The structure of the gene for protein C, an anticoagulant serine protease, was analyzed in 29 unrelated patients with hereditary thrombophilia and protein C deficiency. Gene deletion(s) or gross rearrangement(s) was not demonstrable by Southern blot hybridization to cDNA probes. However, two unrelated patients showed a variant restriction pattern after Pvu II or BamHI digestion, due to mutations in the last exon: analysis of their pedigrees, including three or seven heterozygotes, respectively, with approximately 50% reduction of both enzymatic and antigen level, showed the abnormal restriction pattern in all heterozygous individuals, but not in normal relatives. Cloning of protein C gene and sequencing of the last exon allowed us to identify a nonsense and a missense mutation, respectively. In the first case, codon 306 (CGA, arginine) is mutated to an inframe stop codon, thus generating a new Pvu II recognition site. In the second case, a missense mutation in the BamHI palindrome (GGATCC----GCATCC) leads to substitution of a key amino acid (a tryptophan to cysteine substitution at position 402), invariantly conserved in eukaryotic serine proteases. These point mutations may explain the protein C-deficiency phenotype of heterozygotes in the two pedigrees.

Origin of new mutations in Duchenne muscular dystrophy.

Nine unrelated pedigrees in which Duchenne muscular dystrophy (DMD) was not present in more than one sibship were studied, using 6 DNA polymorphisms closely linked to the DMD gene. The reconstruction of grandparental haplotypes indicates the occurrence of at least three new mutations, two in grandpaternal chromosomes and one in a grandmaternal chromosome. Two additional (but less well documented) new mutations might have occurred respectively in a grandfather's and in a grandmother's chromosome, the latter being represented by a deletion mutation. The new mutations detected in this study therefore add to a total of either three or five out of nine apparently independent mutations present in pedigrees without recurrence of the disorder.

Mapping through somatic cell hybrids and cDNA probes of protein C to chromosome 2, factor X to chromosome 13, and alpha 1-acid glycoprotein to chromosome 9.

The previously unassigned gene coding for the anti-coagulatory protein C has been mapped on chromosome 2 using a cDNA probe and genomic blots from a human-hamster somatic cell hybrid panel. The assignments of the genes coding for the coagulation factor X to chromosome 13, and for alpha 1-acid glycoprotein to chromosome 9 have been confirmed using a similar direct approach.