Stathmin in Cell Proliferation and Cancer Progression.

The phosphoprotein stathmin exerts profound influences on cell proliferation, differentiation and in cell motility. These phenotypic features are displayed in response to specific signals imparted to the cell by biological response modifiers. Stathmin functions as a focal point in co-ordinating and directing the cellular signals into specific and defined pathways. Two biological features that characterise cancer are the deregulation of cell proliferation leading to tumour growth and invasive behaviour. Stathmin is up-regulated in many neoplasms and the modulation of its expression correlates with invasion and metastasis and highly proliferating normal tissues. The integrity of the transduction of extracellular signals is essential for the normal functioning of the cellular machinery in cell differentiation, morphogenesis and cell proliferation, apoptosis, growth and senescence. Stathmin mediates these pathways of signalling. Stathmin has been implicated in both G1-S and G2-M checkpoint control of cell cycle progression by influencing the dynamics of microtubule formation and progression of the cell cycle. Stathmin appears to exert its regulatory effects at both G1-S and G2-M checkpoints by interacting with other cell cycle control proteins such as p53 and rb and with cancer metastasis promoting or inhibiting genes as well as other proteins such as heat shock proteins. Stathmin co-ordinates the signalling by extracellular matrix proteins, and defines intercellular adhesion and cell motility. Therefore, the deregulation of stathmin function would have profound implications in the pathogenesis and progression of cancer.

Prediction of nodal spread of breast cancer by using artificial neural network-based analyses of S100A4, nm23 and steroid receptor expression.

The expression of tumour promoter gene S100A4, metastasis suppressor gene nm23, oestrogen and progesterone receptors, and tumour grade and size have been investigated for their potential to predict breast cancer progression. The molecular and cellular data have been analysed using artificial neural networks to determine the potential of these markers to predict the presence of metastatic tumour in the regional lymph nodes. This study shows that tumour grade and size are poor predictors. The relative expression of S100A4 and nm23 genes is the single most effective predictor of nodal status. Inclusion of oestrogen- and progesterone-receptor status with tumour grade and size markers improves prediction; however, there may be some overlap between steroid receptors and molecular markers. This study also underscores the power of artificial neural network techniques to predict the potential of primary breast cancers to spread to axillary lymph nodes. This could aid the clinician in determining whether invasive procedures of axially node dissection can be obviated and whether conservative forms of treatment might be appropriate in the management of the patient.

Stathmin is involved in S100A4-mediated regulation of cell cycle progression.

S100A4 is a cell proliferation- and cancer metastasis-related gene. Previous studies have shown that over-expression of S100A4 drives the cells into the S-phase of the cell cycle, with concomitant enhancement of p53 detection. This has led to the postulate that S100A4 could be controlling cell cycle progression by sequestering p53 and abrogating its G1-S checkpoint control. Cells induced by S100A4 to enter the S-phase do successfully negotiate the G2-M checkpoint control. Here we show that S100A4 is also involved in the regulation of control at this checkpoint. Stathmin is known to be associated, together with p53 in controlling G2-M transition. We present evidence that the expression of S100A4 and stathmin genes is up regulated in exponentially growing HeLa cells. They are down regulated in parallel when cell proliferation is inhibited by hyperthermia and 4-hydroxynonenal (4-HNE). We postulate that S100A4 might directly induce stathmin up regulation to enable cells to enter into mitosis. Since wild-type p53 is known to down regulate stathmin expression, we further postulate this might also involve S100A4-mediated sequestration of p53. The expression of heme oxygenase (HO-1), a stress-response protein, has been used to monitor effects of hyperthermia, 12-O-tetradecanoly phorbol 13-acetate (TPA) and 4-HNE. All these treatments induced HO-1 and also when cells growing in serum-deficiency were restored with full serum. HO-1 induction occurred irrespective of S100A4 expression status. HO-1 gene has responsive elements for many angiogenic agents and induces marked neovascularisation of tumours. We suggest therefore that S100A4 may not possess angiogenic properties.

Synthesis of (E)-9-(1-pyrenyl)-4-hydroxynon-2-enal, a fluorescent probe of the (E)-4-hydroxynon-2-enal with retained biological properties.

(E)-9-(1-pyrenyl)-4-hydroxynon-2-enal (FHNE), a fluorescent probe of (E)-4-hydroxynon-2-enal (HNE) is synthesised in seven steps and in 35% overall yield, starting from commercially available 1-pyrencarboxyaldehyde. When incubated with cultured HeLa cells this fluorescent probe penetrates cells and particularly concentrates in the region surrounding the nucleus. As the parent compound, HNE it is able to induce the activation of heat shock factor (HSF) and is able to induce the binding of HSF to heat shock element (HSE).

Expression of metastasis-associated genes h-mts1 (S100A4) and nm23 in carcinoma of breast is related to disease progression.

The murine 18A2/mts1 and its human homolog h-mts1 (S100A4), encoding a Ca2+-binding protein belonging to the S-100 family, are associated with high invasive and metastatic potentials of murine tumors, human tumor cell lines in vitro, and human tumors growing as xenografts. The nm23 is a putative metastasis-suppressor gene whose expression has been found to correlate inversely with the metastatic potential of some forms of human cancer. The products of both human genes alter cytoskeletal dynamics, with antagonistic effects. In view of the equivocal association of nm23 with the metastatic potential of human cancer, we suspected that the relative expression of h-mts1 and nm23 might reflect tumor progression more accurately than either of them alone. We describe here the expression of these genes in infiltrating ductal carcinomas of the breast and show that high h-mts1 expression is associated with metastatic spread to the regional lymph nodes. The expression of nm23 on its own did not show a statistically significant inverse correlation with nodal spread. However, the expression status of the two genes, taken together, correlated strongly with the occurrence of nodal metastases. Breast cancers with no detectable expression of h-mts1 were found to be estrogen and progesterone receptor positive. Expression of h-mts1 was not related to tumor differentiation. The clinical data, together with the state of expression of steroid receptors and the expression levels of h-mts1 and nm23 genes, were analyzed using artificial neural networks for accuracy in predicting nodal spread of the carcinomas. These analyses support the conclusion that, overall, h-mts1 expression appears to be associated with and indicative of more aggressive disease. Complemented with nm23, h-mts1 could provide a powerful marker of breast cancer prognosis.

Heat shock modulates the expression of the metastasis associated gene MTS1 and proliferation of murine and human cancer cells.

Mts1 is a metastasis-associated gene of the S-100 gene family and codes for a Ca2+-binding protein. It is highly expressed in murine and human cancers of high invasive and metastatic potential. Recent work has shown that the mts1 protein might be involved in cell cycle regulation. An upregulation of its expression drives cells into the S phase, together with an enhanced expression of p53 phosphoprotein, which has led to the suggestion that mtsl protein might be sequestering p53 thereby abrogating the G1-S checkpoint control normally exerted by p53. Preliminary studies showed that expression of mts1 is downregulated by hyperthermia. We present evidence that in murine BL6 melanoma cells and human HUT cells that hyperthermia downregulates the mts1 gene. It is also downregulated in heat-resistant variants of the B16 melanoma and HUT cells. In parallel, there is a decrease in the size of the S phase fraction and an increase in the doubling time of cells. Cell subjected to hyperthermia show an 2- to 3.5-fold increase in the expression of HSP28 which has been shown to possess a proliferation inhibitory action. It is postulated that a complete regulatory loop involving mtsl, p53, and HSP28 might be involved in cell proliferation.

Characterization of a splice variant of metastasis-associated h-mts1 (S100A4) gene expressed in human infiltrating carcinomas of the breast.

The h-mts1 (S100A4) is a member of the S100 gene family, coding for a calcium-binding protein. It is a metastasis-associated gene whose expression shows strong correlation with the proliferative potential and invasive and metastatic ability of cancers. In a proportion of human intraductal carcinomas of the breast, a shorter variant h-mts1 transcript (h-mts1v) of approximately 450 nucleotides is expressed. We have characterized the transcript using reverse transcriptase-polymerase chain reaction employing exon-specific oligonucleotides. We show here that the noncoding exon 1a/1b is lost in the variant cDNA. Exons 2 and 3, which code for the protein, seem to be present in the variant isoform. The RT-PCR products obtained using exons 2- and 3-specific oligonucleotides showed a high degree of sequence homology with exons 2 and 3 of the h-mts1 gene. The expression of the variant transcript could be influencing disease progression, albeit not as effectively as the normal unspliced h-mts1 transcript.

Structural requirements of aldehydes produced in LPO for the activation of the heat-shock genes in HeLa cells.

The ability of various aldehydes, some of which are produced in lipid peroxidation, to effect heat-shock gene expression and heat-shock proteins synthesis was evaluated in HeLa cells. Only (E)-4-hydroxyalk-2-enals were active both in racemic and homochiral form. Between the reported primary metabolic products of (E)-4-hydroxynon-2-enal, only the glutathione conjugates were active, whereas (E)-4-hydroxynon-2-enoic acid and 2-nonen-1,4-diol were inactive. Also, unnatural (E)-5-hydroxynon-2-enal and (E)-5-hydroxyhex-2-enal were active, whereas (E)-6-hydroxynon-2-enal was inactive. Thus, it was established that the active aldehydic compounds must possess an (E)-2 double bond and an hydroxy group in a position suitable for the formation of a cyclic hemiacetal in a possible adduct of these aldehydes with proteins. An irreversible binding to proteins could be the first step of the mechanism by which these compounds exert their biological activity.

Metastasis-associated mts1 gene expression is down-regulated by heat shock in variant cell lines of the B16 murine melanoma.

Tumour cells are more heat sensitive than corresponding normal cells but the reasons for this are poorly understood. Here we report that induction of heat shock proteins was associated with a down-regulation of the metastasis associated mts1 gene in BL6-B16 murine melanoma cells, and the heat-resistant HTG variant of the BL6 line. Melanocyte stimulating hormone, which does not affect B16 cell proliferation but upregulates mts1 expression, only marginally enhanced heat shock protein expression in F1 cells as determined by immunohistochemical methods. Retinoic acid, which inhibits cell proliferation and down-regulates the mts1 gene, reduced heat shock protein expression in the ML8-B16 variant line. This suggests that the changes in the heat shock protein expression reported here may be cell proliferation related. Heat shock proteins are known to stabilize microtubules, whereas mts1 has been implicated in their depolymerization. Taxol, which stabilizes microtubules and arrests cells at the G1 phase of the cell cycle, down-regulated mts1 gene expression in both F1 and ML8 lines. Taxol also reduced heat shock protein expression in ML8 cells. These data suggest opposing functions of heat shock proteins and the mts1 gene in microtubule polymerization, and may provide a rationale for the use of hyperthermia as a treatment for tumours.

In vitro activation of heat shock transcription factor by 4-hydroxynonenal.

In the activation of eukaryotic heat shock genes, the acquisition of a binding ability to specific DNA sequence by a transcriptional activator, heat shock factor (HSF), is believed to be a crucial step. The induction of this new DNA binding activity of HSF is also obtained in a cell-free system (in vitro activation) by hyperthermia or at physiological temperature by calcium ions, low pH, urea, or non-ionic detergent. We report here the in vitro activation of HSF by treating at 0 degrees C a HeLa cell-free system with the aldehyde 4-hydroxynonenal (HNE), a highly cytotoxic product of lipid peroxidation. The in vitro activation of HSF by HNE occurred only if some components of the cell-free system were not sedimented at 100,000 x g. The reason for this is unclear but the release of active HSF from nuclei of unshocked cells and the involvement of Ca2+ contained in the mitochondria and ER have been excluded. Although HNE is known to be a sulfhydryl blocking agent, the results obtained with N-ethylmaleimide suggest that different mechanisms might be involved in the in vitro activation of HSF by HNE.

4-Hydroxynonenal induces a DNA-binding protein similar to the heat-shock factor.

By using a gel mobility assay, we have shown that treatment of HeLa cells with 4-hydroxynonenal, a major product of the peroxidation of membrane lipids and an inducer of heat-shock proteins, has the same effect as heat shock in causing the appearance of a protein which binds to the sequence of DNA specific for the induction of heat-shock genes. Lipoperoxidation and heat exposure seem to share a common mechanism of specific gene activation.

The action of 4-hydroxynonenal on heat shock gene expression in cultured hepatoma cells.

Hep G2 cells do not synthesize heat-shock proteins when incubated with ADP-iron, under conditions that can trigger lipoperoxidation. However, exposure of these cells to added 4-hydroxynonenal (HNE), one of the main products of lipoperoxidation, induces the synthesis of hsp70, the most conserved among heat-shock proteins. HNE acts mainly on transcription: in Hep G2 cells the increase in the steady-state level of hsp70 mRNA is detectable by two different hybridization techniques.

C-myc gene expression in heat-adapted and heat-shocked cells.

The steady-state level of c-myc transcripts increases in cells exposed to high temperatures. Therefore c-myc can be included with c-fos in the family of heat-inducible oncogenes. Activation of c-myc upon heat exposure could in turn account for the induction of heat-shock proteins but recent observations suggest also alternative interpretations.

Oxidative stress induces a subset of heat shock proteins in rat hepatocytes and MH1C1 cells.

Lipoperoxidative damage caused by exposure of isolated hepatocytes or cultivated hepatoma cells to ADP-iron or to 4-hydroxynonenal induces the synthesis of some proteins which are different under these two conditions but are always a subset of the proteins induced in each type of cells upon heat-shock (heat-shock proteins). For at least one of these proteins (hsp 31), induced by 4-hydroxynonenal, the increase is dose-dependent and the effect of heat and the chemical seems to be additive. Lipoperoxidation may be implicated in the induction of some of the heat shock proteins, but reproduces only incompletely the response of protein synthesis typical of heat-shock conditions.

Synthesis of heat-shock proteins and tumor growth.

Exposure to hyperthermia induces the preferential synthesis of a set of proteins, known as heat-shock proteins. The synthesis of heat-shock proteins has been studied in rat liver cells, and human lymphocytes, and in their neoplastic counterparts. Tumor cells synthesize heat-shock proteins essentially as their normal controls, but the response of ascites hepatoma cells depends on the presence of glucose in the medium. Solid hepatoma slices seem to synthesize some heat-shock proteins constitutively, i.e., before exposure to high temperature. Any possible interpretation of this fact must take into account the growth of tumor cells.

Soluble factors of protein synthesis in rat liver during the acute-phase reaction.

Cell sap of liver cells from rats undergoing an acute-phase reaction has an increased capacity for binding leucine to tRNA. This increased capacity does not depend on concurrent changes in the leucine pool. The kinetics of activity of leucyl-tRNA synthetase from acute-phase cell sap do not show relevant differences from the normal. Acute-phase cell sap contains more tRNA than its normal counterpart. Experiments performed with increasing amounts of exogenous deacylated tRNA demonstrate that under our experimental conditions the observed higher concentration of tRNA in acute-phase cell sap could explain the increased activity in leucine incorporation into leucyl-tRNA.

The role of nuclei, polyribosomes and cytosol factors in the onset of the acute-phase reaction in the liver cell.

Nuclei isolated from livers of turpentine-treated rats show an increased RNA synthesis, reaching a maximum at 10 h after treatment. The stimulation affects both alpha-amanitin-resistant and alpha-amanitin-sensitive activities, suggesting that pre-ribosomal and pre-messenger RNA formation are activated at the same time and to the same extent. The amount of ribosomal RNA, which is still normal 10 h after treatment, increases significantly at 24 h, but the increase is limited to the bound ribosomes, in keeping with the fact that the acute phase reactants are export proteins. These ribosomes, however, are not more active per se and the stimulation of protein synthesis in cell-free preparations depends essentially on an increased activity of soluble factors located in the cytosol. In living cells these soluble factors co-operate with an increased amount of some specific mRNAs and an expanded population of membrane-bound polyribosomes, thus leading to the increased protein synthesis peculiar to the liver of turpentine-treated rats.

The effect of phenobarbital on protein metabolism of liver. Results with isolated hepatocytes.

A technique has been devised which makes use of an amino acid alternatively labeled with either [14C] or [3H], and permits the simultaneous evaluation of synthesis, catabolism and secretion by the same sample of isolated rat liver cells during the same time-period of incubation. This technique has been used to study protein metabolism of liver cells isolated from rats treated with 4 doses of phenobarbital (8 mg/100 g body weight) given in the 4 days before killing the animals. Total protein synthesis and secretion do not change in phenobarbital-treated rats; albumin represents 40% of secreted protein in both normal and treated rats. On the contrary, the parameters which indicate protein degradation are lower in phenobarbital-treated than in normal rats, showing that protein catabolism is appreciably reduced in the liver cells obtained from rats treated with phenobarbital.

Thioacetamide effects on protein metabolism in the liver: lessons from isolated hepatocytes.

The effects of a short-term treatment with thioacetamide have been studied in isolated hepatocytes obtained from intoxicated rats. A technique has been developed which utilizes leucine alternatively labeled with either [14C] or [3H] and permits the simultaneous evaluation of protein synthesis, catabolism and secretion in the same cell during the same incubation period. The results indicate that short-term thioacetamide treatment causes an overall slowing-down of protein metabolism. Protein synthesis, however, decreases less than protein degradation and total protein secretion; albumin secretion, which is also less than normal, seems to be less compromised than total protein secretion.