Hydroxytyrosol, a natural molecule occurring in olive oil, induces cytochrome c-dependent apoptosis.

2-(3,4-Dihydroxyphenyl)ethanol (DPE), a naturally occurring phenolic antioxidant molecule found in olive oil, has been reported to exert several biological and pharmacological activities. We studied the effect of this compound on the proliferation and survival of HL60 cell line. Concentrations from 50 to 100 microM DPE, comparable to its olive oil content, caused a complete arrest of HL60 cell proliferation and the induction of apoptosis. This was demonstrated by flow cytometric analyses, poly(ADP-ribose) polymerase cleavage, and caspase 3 activation. The apoptotic effect requires the presence of two ortho-hydroxyl groups on the phenyl ring, since tyrosol, 2-(4-hydroxyphenyl)ethanol, did not induce either cell growth arrest or apoptosis. DPE-dependent apoptosis is associated with an early release of cytochrome c from mitochondria which precedes caspase 8 activation, thus ruling out the engagement of cell death receptors in the apoptotic process. 2-(3,4-Dihydroxyphenyl)ethanol induced cell death in quiescent and differentiated HL60 cells, as well as in resting and activated peripheral blood lymphocytes, while did not cause cell death in two colorectal cell lines (HT-29 and CaCo2). These results suggest that DPE down-regulates the immunological response, thus explaining the well-known antinflammatory and chemopreventive effects of olive oil at the intestinal level.

p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation: evidence of a decreased proteasome-dependent degradation.

Development of human neuroblastoma is due to an arrest in the differentiation program of neural crest sympathoadrenal progenitor cells. However, neuroblastomas, as well as their derived cell lines, maintain the potentiality of terminal differentiation. We investigated the molecular mechanisms by which retinoic acid, a molecule introduced in clinical trials for chemotherapy, induces differentiation in neuroblastoma cell lines. Our findings demonstrate that the retinoic acid-dependent growth arrest of LAN-5 neuroblastoma cell line is associated to a very large accumulation (>tenfold) of p27Kip1 protein, a cyclin-dependent kinase inhibitor; the protein binds and inhibits cyclin-dependent kinase 2, 4 and 6 activities, thus hampering pRb and p107 phosphorylation. p27Kip1 build-up was observable as an early phenomenon (12 - 24 h) after retinoic exposure and resulted in a time-dependent accumulation of high quantities of a free p27Kip1 form. Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27kip1. No noticeable variation of other components of G1 phase cell cycle engine was observed. Pulse-chase experiments showed a remarkable elongation of p27Kip1 half-life in retinoic-treated LAN-5, while no enhancement of p27Kip1 gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27Kip1 was sensitive to two highly specific proteasome inhibitors, LLnL and lactacystin, while the calpain inhibitor II ALLM and the cysteine protease inhibitor E64 did not modify the level of the protein. LLnL treatment caused a very rapid (2 h) build-up of the Cdk inhibitor content and the accumulation of higher molecular weight anti-p27Kip1 immunoreactive bands, which probably represent ubiquitinated forms of the protein. Finally, in vitro experiments demonstrated that extracts prepared from retinoic-treated LAN-5 cells degraded recombinant p27Kip1 at a rate remarkably slower than the untreated cells. Our results indicate that retinoic acid strongly increases p27Kip1 levels by down-regulating the ubiquitin-proteasome p27Kip1 degrading pathway.

Resveratrol arrests the cell division cycle at S/G2 phase transition.

Resveratrol (3,5,4'-trihydroxystilbene) is a naturally occurring phytoalexin, found in grapes and wine, which has been reported to exert a variety of important pharmacological effects. We have investigated the activity of resveratrol on proliferation and differentiation of the promyelocitic cell line HL-60. A concentration as low as 30 microM causes a complete arrest of proliferation and a rapid induction of differentiation towards a myelo-monocytic phenotype. Analyses by flow cytometry showed the absence of the G2/M peak and the accumulation of cells in G1 and S phases. Moreover, at the concentrations employed, a very low amount of apoptotic cells was evidenced. A detailed biochemical analysis demonstrated that the G1 phase of the cell division cycle engine was completely unmodified by resveratrol addition, thus indicating that the G1 --> S transition occurs normally. Conversely, after only 24 h treatment, a significant increase of cyclins A and E could be observed along with the accumulation of cdc2 in the inactive phosphorylated form. These data demonstrate that resveratrol causes a complete and reversible cell cycle arrest at the S phase checkpoint.

Structural and functional analysis of cyclin-dependent kinase inhibitor genes (CDKN2A, CDKN2B, and CDKN2C) in neuroblastoma.

The status of the CDKN2A gene family, including CDKN2A, CDKN2B, and CDKN2C, was investigated in 24 cases of neuroblastoma. These genes were selected on the basis of 1) high incidence of their inactivation in several human cancers and 2) their localization on chromosomal regions (9p and 1p) frequently rearranged in neuroblastomas. Detailed molecular analyses indicated the absence of homozygous deletions and point mutations involving these genes in all investigated tumor samples. However, when loss of heterozygostity for chromosome 9p21 (the region where CDKN2A and CDKN2B are localized) was investigated, 16% of cases showed abnormalities in an area telomeric to the CDKN2A locus. To study transcriptional silencing of the CDKN2A gene, the methylation status of exon 1 was examined. In about 35% of cases, a partial methylation was evidenced. Analysis of the CDKN2A mRNA expression, however, did not show any relationship between methylation status and gene transcription. Finally, expression of the CDKN2B gene was demonstrated in all stage IV neuroblastomas, whereas none of stage I tumors expressed this gene. This finding suggests the occurrence of a correlation between CDKN2B transcription and tumor phenotype.

Inactivation of cyclin-dependent kinase inhibitor genes and development of human acute leukemias.

A large body of evidence has definitely demonstrated that cancer development and/or progression is strictly linked to alterations of molecular mechanisms controlling the cell division cycle. In particular, those aberrations which cause a shortening of G1 phase length and a deregulated S phase entry seem to be very important. Two main tumor suppressor loci, involved in the cell cycle regulation, are frequently altered in human tumors. One is located on 13q14 chromosome and includes the gene coding pRb protein while the other is located on 9p21 chromosome and involves two genes, namely p16INK4A and p15INK4B which belong to the same gene family. While RB1 gene is scarcely altered in hematological tumors, the putative tumor suppressor gene(s) on 9p21 appear(s) to be frequently inactivated in some subtypes of cancers derived from hematopoietic tissues. This manuscript will review the main biochemical aspects of the cell division cycle with major emphasis devoted to the findings regarding the recently characterized small proteic mitotic inhibitors and to their possible role in cancer formation. Particular attention will be paid to the data concerning the incidence of p16INK4A (and p15INK4B) gene(s) inactivation in human acute lymphoblastic leukemias. Indeed, such gene(s) seems to be the main, and until now the unique, tumor suppressor gene consistently altered in this acute hematological cancer diseases. Finally, future directions in studies on the connection between cell cycle control and leukemogenesis will be analyzed.

Biochemical characterization of p16INK4- and p18-containing complexes in human cell lines.

The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G1 cell cycle phase. One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs. We have evaluated the amount of two such inhibitors, namely p16(INK4) and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins. The results obtained indicated that (i) p18 gene expression, unlike p16(INK4) gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16(INK4) and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase. Moreover, employing the human native partially purified p16(INK4)or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16(INK4) bimolecular complex. Our findings suggest that during the cell division cycle the members of the p16(INK4) protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G1 --> S transition.

High-performance liquid chromatographic separation of natural adenosyl-sulphur compounds.

A rapid, sensitive and specific high-performance liquid chromatographic method for the simultaneous separation of natural adenosyl-sulphur compounds has been developed. The compounds were separated by using the strong cation-exchange resin Partisil 10 SCX with isocratic elution. The adenosyl-compounds were monitored by an ultraviolet detector operating at 254 nm. Sensitivity was greater than 50 pmoles for all compounds tested and standard curves were found to be linear for concentrations of up to 50 nmoles. The method can be applied to biological samples for the estimation of S-adenosyl-L-methionine,S-adenosyl-L-homocysteine and S-adenosyl-(5')-3-methyl-thiopropylamine and for measurement of enzyme activities involving the above-mentioned compounds.