What is the restriction point?

The restriction point (R) separates two functionally different parts of G1 in continuously cycling cells. G1-pm represents the postmitotic interval of G1 that lasts from mitosis to R. G1-ps represents the pre S phase interval of G1 that lasts from R to S. G1-pm is remarkably constant in length (its duration is about three hours) in the different cell types studied so far. G1-ps, however, varies considerably, indicating that entry into S is not directly followed after passage through R. Progression through G1-pm requires continuous stimulation by mitogenic signals (e.g. growth factors) and a high rate of protein synthesis. Interruption of the mitogenic signals or moderate inhibition of protein synthesis leads to a rapid exit from the cell cycle to G0 in normal (untransformed) cells. Upon restimulation with mitogenic signals, the cell returns to the same point in G1-pm from which it left the cell cycle. Thus the cell seems to have a memory for how far it has advanced through G1-pm, suggesting that a continuous structural alteration, for example chromatin decondensation, takes place in G1. The molecular background to transition from growth factor dependence in G1-pm to growth factor independence in G1-ps (a switch which represents commitment to a new cell cycle and passage through R) is still not fully understood. Cyclin-dependent kinase (cdk)-mediated hyperphosphorylation of the retinoblastoma protein (Rb), and concomitant liberation (and activation) of members of the E2F family of transcription factors, are probably important aspects of R control in normal cells. A key component here could be cdk2 activity which is controlled by cyclin E. When cdk2 activity starts to increase rapidly in G1, due to activation of a positive feedback loop, it reaches a critical level above which cdk inhibitors (CKIs) such as p21 and p27 are outweighed; the cell has then become independent of mitogenic and inhibitory signals and is committed to a new cell cycle. However, other components are probably also involved in R control. For instance, a 'cryptic' R (a G1-pm-like state) can be induced even in tumour cells that do not respond to growth factor starvation or protein synthesis inhibitors, and are therefore probably defective in the cdk-Rb-E2F pathway. Possibly, a certain degree of chromatin decondensation has to take place after mitosis in order to allow transcription of, for example, the cyclin E gene or other critical E2F targets. Although the molecular basis for restriction point control still remains unclear, we can expect rapid progress in this important field over the next few years.

Increased activity of L-type Ca2+ channels exposed to serum from patients with type I diabetes.

Type I diabetes [insulin-dependent diabetes mellitus (IDDM)] is an autoimmune disease associated with the destruction of pancreatic beta cells. Serum from patients with IDDM increased L-type calcium channel activity of insulin-producing cells and of GH3 cells derived from a pituitary tumor. The subsequent increase in the concentration of free cytoplasmic Ca2+ ([Ca2+]i) was associated with DNA fragmentation typical of programmed cell death or apoptosis. These effects of the serum were prevented by adding a blocker of voltage-activated L-type Ca2+ channels. When the serum was depleted of immunoglobulin M (IgM), it no longer affected [Ca2+]i. An IgM-mediated increase in Ca2+ influx may thus be part of the autoimmune reaction associated with IDDM and contribute to the destruction of beta cells in vivo.

Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate.

Inositol hexakisphosphate (InsP6), the dominant inositol phosphate in insulin-secreting pancreatic beta cells, inhibited the serine-threonine protein phosphatases type 1, type 2A, and type 3 in a concentration-dependent manner. The activity of voltage-gated L-type calcium channels is increased in cells treated with inhibitors of serine-threonine protein phosphatases. Thus, the increased calcium channel activity obtained in the presence of InsP6 might result from the inhibition of phosphatase activity. Glucose elicited a transient increase in InsP6 concentration, which indicates that this inositol polyphosphate may modulate calcium influx over the plasma membrane and serve as a signal in the pancreatic beta cell stimulus-secretion coupling.

PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Insulin-like growth factor-1 receptor acts as a growth regulator in synovial sarcoma.

Synovial sarcomas account for 5-10% of all soft tissue sarcomas and the majority of synovial sarcomas display characteristic t(X;18) translocations that result in enhanced transcription of the insulin-like growth factor-2 (IGF-2) gene. IGF-2 is an essential fetal mitogen involved in the pathogenesis of different tumours, leading to cellular proliferation and inhibition of apoptosis. Here we asked whether activation of IGF signalling is of functional importance in synovial sarcomas. We screened human synovial sarcomas for expression of IGF-2 and the phosphorylated IGF-1 receptor (IGF-1R), which mainly mediates the proliferative and anti-apoptotic effects of IGF-2. Since both the phosphatidylinositol 3'-kinase (PI3K)-AKT pathway and the MAPK signalling cascade are known to be involved in the transmission of IGF-1R signals, expression of phosphorylated (p)-AKT and p-p44/42 MAPK was additionally assessed. All tumours expressed IGF-2 and 78% showed an activated IGF-1R. All tumours were found to express p-AKT and 92% showed expression of activated p44/42 MAPK. To analyse the functional and potential therapeutic relevance of IGF-1R signalling, synovial sarcoma cell lines were treated with the IGF-1R inhibitor NVP-AEW541. Growth was impaired by the IGF-1R antagonist, which was consistently accompanied by a dose-dependent reduction of phosphorylation of AKT and p44/42 MAPK. Functionally, inhibition of the receptor led to increased apoptosis and diminished mitotic activity. Concurrent exposure of selected cells to NVP-AEW541 and conventional chemotherapeutic agents resulted in positive interactions. Finally, synovial sarcoma cell migration was found to be dependent on signals transmitted by the IGF-1R. In summary, our data show that the IGF-1R might represent a promising therapeutic target in synovial sarcomas.

The insulin-like growth factor-1 receptor inhibitor PPP produces only very limited resistance in tumor cells exposed to long-term selection.

The cyclolignan PPP was recently demonstrated to inhibit the activity of insulin-like growth factor-1 receptor (IGF-1R), without affecting the highly homologous insulin receptor. In addition, PPP caused complete regression of xenografts derived from various types of cancer. These data highlight the use of this compound in cancer treatment. However, a general concern with antitumor agents is development of resistance. In light of this problem, we aimed to investigate whether malignant cells may develop serious resistance to PPP. After trying to select 10 malignant cell lines, with documented IGF-1R expression and apoptotic responsiveness to PPP treatment (IC50s less than 0.1 microM), only two survived an 80-week selection but could only tolerate maximal PPP doses of 0.2 and 0.5 microM, respectively. Any further increase in the PPP dose resulted in massive cell death. These two cell lines were demonstrated not to acquire any essential alteration in responsiveness to PPP regarding IGF-1-induced IGF-1R phosphorylation. Neither did they exhibit any increase in expression of the multidrug resistance proteins MDR1 or MRP1. Consistently, they did not exhibit decreased sensitivity to conventional cytostatic drugs. Rather, the sensitivity was increased. During the first half of the selection period, both cell lines responded with a temporary and moderate increase in IGF-1R expression, which appeared to be because of an increased transcription of the IGF-1R gene. This increase in IGF-1R might be necessary to make cells competent for further selection but only up to a PPP concentration of 0.2 and 0.5 microM. In conclusion, malignant cells develop no or remarkably weak resistance to the IGF-1R inhibitor PPP.

Bactericidal antisense effects of peptide-PNA conjugates.

Antisense peptide nucleic acids (PNAs) can specifically inhibit Escherichia coli gene expression and growth and hold promise as anti-infective agents and as tools for microbial functional genomics. Here we demonstrate that chemical modification improves the potency of standard PNAs. We show that 9- to 12-mer PNAs, especially when attached to the cell wall/membrane-active peptide KFFKFFKFFK, provide improvements in antisense potency in E. coli amounting to two orders of magnitude while retaining target specificity. Peptide-PNA conjugates targeted to ribosomal RNA (rRNA) and to messenger RNA (mRNA) encoding the essential fatty acid biosynthesis protein Acp prevented cell growth. The anti-acpP PNA at 2 microM concentration cured HeLa cell cultures noninvasively infected with E. coli K12 without any apparent toxicity to the human cells. These results indicate that peptides can be used to carry antisense PNA agents into bacteria. Such peptide-PNA conjugates open exciting possibilities for anti-infective drug development and provide new tools for microbial genetics.

Silanized nucleic acids: a general platform for DNA immobilization.

We have developed a method for simultaneous deposition and covalent cross-linking of oligonucleotide or PCR products on unmodified glass surfaces. By covalently conjugating an active silyl moiety onto oligonucleotides or cDNA in solutions we have generated a new class of modified nucleic acids, namely silanized nucleic acids. Such silanized molecules can be immobilized instantly onto glass surfaces after manual or automated deposition. This method provides a simple and rapid, yet very efficient, solution to the immobilization of prefabricated oligonucleotides and DNA for chip production.

Kinetics of G1 progression in 3T6 and SV-3T3 cells following treatment by 25-hydroxycholesterol.

The kinetics of cell cycle progression in continuously proliferating 3T3 fibroblasts and two tumor transformed derivatives (3T6 and SV 3T3 cells) following treatment by growth-factor deprivation (serum starvation) or 25-hydroxycholesterol were studied. Normal 3T3 cells were found to respond immediately (in the first cycle) to growth factor deprivation by leaving the cell cycle from G1, whereas the tumor transformed derivatives did not. However, all three cell types were forced to stop the progression through the beginning of G1 when treated by 25-hydroxycholesterol. It was ensured that the doses of 25-hydroxycholesterol used really induced substantial decrease of HMG CoA reductase activity. However, the effects of serum starvation on HMG CoA reductase activity varied considerably. In 3T3 cells HMG CoA reductase activity was substantially depressed, in 3T6 cells it was moderately depressed, and in SV-3T3 cells it was not depressed at all. This difference of HMG CoA reductase activity between 3T6 and SV-3T3 cells was related to the difference of growth activity in serum-free medium. The data indicate that a certain activity of HMG CoA reductase is required for the proliferation of normal as well as tumor transformed cells but also that impairment of the control of HMG CoA reductase, leading to increased enzyme activity, may result in uncontrolled growth in tumor transformed cells.

Mevalonic acid products as mediators of cell proliferation in simian virus 40-transformed 3T3 cells.

Effects of treatment with serum-free medium and 25-hydroxycholesterol (25-OH) on the cell cycle of simian virus 40-transformed 3T3 fibroblasts, designated SV-3T3 cells, were studied and compared with simultaneous effects on the activity of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase and incorporation of [3H]mevalonic acid into cholesterol, Coenzyme Q, and dolichol. The data confirm our previous finding (O. Larsson and A. Zetterberg, Cancer Res., 46: 1233-1239, 1986) that 25-OH inhibits the cell cycle traverse of SV-3T3 cells specifically in early G1. In contrast, treatment with serum-free medium had no effect on cell cycle progression. The effect of 25-OH on the cell cycle traverse was correlated to a substantial decrease in the activity of HMG CoA reductase, whereas there was no change in the rate of [3H]mevalonic acid incorporated into cholesterol, Coenzyme Q, and dolichol. When the cells were exposed to serum-free medium, there was no depression of activity of HMG CoA reductase, and the rate of [3H]mevalonic acid incorporated into dolichol and cholesterol was not affected in any appreciable degree. In contrast the rate of Coenzyme Q synthesis was substantially decreased as a result of serum depletion. A similar decrease in Coenzyme Q synthesis was also achieved by treating the cells with cholesterol-poor serum. This indicates that the rate of Coenzyme Q synthesis is dependent on the concentration of cholesterol in the culture medium. In order to analyze whether some of the products in the mevalonic acid biosynthetic pathway may be of importance in the control of G1 traverse and cell proliferation of SV-3T3 cells, cholesterol, Coenzyme Q, and dolichol were added as supplements to cells treated with 25-OH. It was shown that dolichol was capable of overcoming the 25-OH-induced inhibition of G1 traverse efficiently, whereas cholesterol and Coenzyme Q were considerably less effective. Considered together with the fact that the activity of HMG CoA reductase and incorporation of mevalonic acid into dolichol were unaffected following serum-free treatment, the results suggest that maintenance of a certain level of de novo synthesis of dolichol may contribute to the capability of SV-3T3 cells to proliferate in serum-free medium.

Inhibition of N-linked glycosylation using tunicamycin causes cell death in malignant cells: role of down-regulation of the insulin-like growth factor 1 receptor in induction of apoptosis.

Recent studies have shown that inhibition of N-linked glycosylation using tunicamycin (TM) induces cell death in cultured cells (O. Larsson et al., J. Cell Sci., 106: 299-307, 1993; J. Y. Chang and V. Korolev, Exp. Neurol., 137: 201-211, 1996). The mechanisms underlying TM-induced cell death seem, however, to be complex in nature. In SV40-transformed cells, TM triggers within a few minutes mechanisms subsequently leading to apoptosis. These mechanisms, although still not fully understood, involve an elevation of [Ca2+]i (M. Carlberg et al., Carcinogenesis, 17: 2589-2596, 1996). In contrast, in melanoma cells, TM has to be present continuously for 24-48 h to elicit apoptosis. Before the appearance of apoptotic melanoma cells, assayed by gel-electrophoretic detection of oligonucleosomally fragmented DNA, the binding of insulin-like growth factor I (IGF-I) to the cells had become drastically decreased, which in turn was found to be due to down-regulation of IGF-I receptor proteins at the cell surface. Incubation of the cells with an antibody (alpha IR-3) against the IGF-I-binding site of the receptor resulted also in apoptosis, the kinetics of which were almost identical to those following treatment with TM. Furthermore, coincubation of a high concentration of IGF-I (50 ng/ml) with TM totally rescued the melanoma cells from apoptotic cell death during the first 48 h after addition of the drugs. This effect was shown to be abolished fully by alpha IR-3. Taken together, our data suggest strongly that N-linked glycosylation plays an important role in maintenance of viability of melanoma cells through regulating the translocation of IGF-I receptor to the cell surface.

Elevated uptake of low density lipoproteins by human lung cancer tissue in vivo.

In order to explore new treatment modalities for cancer, it is important to identify qualitative or quantitative differences in metabolic processes between normal and malignant cells. Previous in vitro and in vivo studies have shown that acute myelogenous leukemia cells have elevated receptor-mediated uptake of low density lipoproteins (LDL), compared to normal WBC. High receptor-mediated uptake of LDL by certain cancer cells in tissue culture and experimental tumors in animals in vivo has also been demonstrated. The present study was undertaken to compare the in vivo assimilation of LDL by human lung cancer tissue with that by surrounding lung tissue. Ten patients with newly diagnosed lung tumors, scheduled for surgery, received an i.v. injection of [14C]sucrose-labeled LDL. Following cellular uptake and degradation of the LDL particle, the radiolabeled sucrose moiety remains trapped in the lysosomal compartment, making this labeling technique useful for in vivo studies of tissue uptake of LDL. Radioactivity was determined in plasma and in tissue biopsies obtained at surgery 1-3 days after injection. The uptake of radioactivity in lung cancer tissue was elevated (1.5-3.0-fold), compared to surrounding tissue, in 7 of 9 patients with primary lung cancer. The most rapid preoperative disappearance of radioactivity from plasma was found in 2 patients with large tumors exhibiting high LDL uptake, relative to normal lung tissue. These findings support the hypothesis that the selectivity of cytotoxic agents can be enhanced also in nonhematological malignancies by administering the drugs incorporated in LDL particles.

Expression of insulin-like growth factor-1 receptor in synovial sarcoma: association with an aggressive phenotype.

It is well known that insulin-like growth factor-1 receptor (IGF-1R) plays a crucial role in proliferation and survival of transformed cells. Overexpression of IGF-1R in certain tumors has been reported, but there is still little known about its importance in vivo. Here, we evaluated the IGF-1R levels in 35 human synovial sarcoma tumors by Western blot and reverse transcriptase-PCR. In 18 of these, IGF-1R was detectable by Western blot, whereas 17 were nondetectable. There was a significant association between the amount of receptor proteins and mRNA transcripts. Furthermore, we found that the IGF-1R Western blot-positive tumors were associated with a high incidence of lung metastases. Eleven of 18 (61%) developed metastases in the IGF-1R detectable group, compared to 3 of 17 (18%) in the nondetectable group (P = 0.01). Moreover, in the detectable group of IGF-1R, 12 of 18 (67%) exhibited a high tumor cell proliferative rate, compared to 5 of 16 (31%) in the nondetectable group (P = 0.04). On the other hand, no association was found between the IGF-1R and type of fusion gene transcript (SYT-SSX1 or SYT-SSX2). Our results suggest that expression of IGF-1R can underlie an aggressive phenotype in synovial sarcoma.

Abolition of mevinolin-induced growth inhibition in human fibroblasts following transformation by simian virus 40.

The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase was higher in SV40-transformed human fibroblasts (90-VA VI) than in normal ones (HDF). In both these cell types mevinolin (25 microM) caused an 85-90% depression of HMG CoA reductase activity and of the incorporation of [3H]acetate into sterols. In HDF this was coupled to an efficient block of cell growth, whereas the growth of 90-VA VI was only slightly reduced by mevinolin. In HDF, mevinolin (25 microM) also abolished essentially all dilichol synthesis, as measured by incorporation of [3H]acetate. In contrast, dolichol synthesis remained unaltered, or was increased, in mevinolin-treated 90-VA VI. We suggest that these different responses of dolichol synthesis may depend on different substrate affinities of the rate-limiting enzyme in the dolichol pathway. However, if 90-VA VI was treated with 25-hydroxycholesterol (25-OH), an alternative inhibitor of HMG CoA reductase, the cellular growth as well as dolichol synthesis was significantly decreased. Since the inhibitory effect of 25OH on HMG CoA reductase activity did not exceed that of mevinolin, it seems that 25-OH, besides HMG CoA reductase, inhibits steps distal to HMG CoA reductase. This notion was further supported by the finding that addition of mevalonate did not prevent the 25-OH-induced growth inhibition. However, if dolichol was added along with 25-OH, the block was partially prevented, indicating that a critical level of de novo synthesis of dolichol for cellular growth.

Increased expression of insulin-like growth factor I receptor in malignant cells expressing aberrant p53: functional impact.

We investigated the functional impact of p53 on insulin-like growth factor I receptor (IGF-IR) expression in malignant cells. Using the BL-41tsp53-2 cell line, a transfectant carrying temperature-sensitive (ts) p53 and endogenous mutant p53 (codon 248), we demonstrated a drastic down-regulation of plasma membrane-bound IGF-IRs on induction of wild-type p53. However, a similar response was obtained by treatment of BL-41tsp53-2 cells expressing mutant ts p53 with a p53 antisense oligonucleotide. Thus, even if the negative effect of wild-type p53 predominates under a competitive condition, these data indicate that mutant p53 may be important for up-regulation of IGF-IR. To further elucidate this issue, three melanoma cell lines (BE, SK-MEL-5, and SK-MEL-28) that overexpressed p53 were investigated. The BE cell line has a "hot spot" mutation (codon 248) and expresses only codon 248-mutant p53. SK-MEL-28 has a point mutation at codon 145. SK-MEL-5 cells did not exhibit any p53 mutations, but the absence of p21Waf1 expression suggested functionally aberrant p53. Our data suggest that interaction with Mdm-2 may underlie p53 inactivation in these cells. Using p53 antisense oligonucleotides, we demonstrated a substantial down-regulation of cell surface expression of IGF-IR proteins in all melanoma cell lines after 24 h. This was paralleled by decreased tyrosine phosphorylation of IGF-IR and growth arrest, and, subsequently, massive cell death was observed (this was also seen in BL-41tsp53-2 cells with mutant conformation of ts p53). Taken together, our results suggest that up-regulation of IGF-IR as a result of expression of aberrant p53 may be important for the growth and survival of malignant cells.

The SYT-SSX1 variant of synovial sarcoma is associated with a high rate of tumor cell proliferation and poor clinical outcome.

Cytogenetically, synovial sarcoma (SS) is characterized by the translocation t(X;18)(p11.2;q11.2), resulting in a fusion between the SYT gene on chromosome 18 and SSX1 or SSX2 on the X chromosome and the formation of new chimeric genes, SYT-SSX1 or SYT-SSX2. We examined the potential clinical relevance of SYT-SSX1 and SYT-SSX2 fusion transcripts together with tumor proliferation. In a series of 33 patients with primary SS, the type of fusion transcript was assessed by reverse transcription-PCR and sequence analysis. The proliferation rate was analyzed using anti-Ki-67 antibodies. One case carrying an atypical transcript with a 57-bp insert was excluded, leaving 13 SYT-SSX1 and 19 SYT-SSX2 cases for analysis. The hazard ratio (with respect to metastasis-free survival for patients with SYT-SSX1 versus patients with SYT-SSX2 fusion transcripts was 7.4 (95% confidence interval, 1.5-36; log-rank P = 0.004). There was also an association with reduced overall survival for patients with SYT-SSX1 compared to patients with SYT-SSX2 (hazard ratio, 8.5; 95% confidence interval, 1.0-73; log-rank P = 0.02). The 5-year metastasis-free survival for patients with SYT-SSX1 was 42% versus 89% for patients with SYT-SSX2. There was a significant association between SYT-SSX1 and a high tumor proliferation rate (P = 0.02). We conclude that the findings suggest that the type of SYT-SSX fusion transcript determines the proliferation rate and is an important predictor of clinical outcome in patients with SS.

Tumor cell survival dependence on the DHX9 DExH-box helicase.

The NTP-dependent DExH/D-box helicase DHX9 is a key participant in a number of gene regulatory steps, including transcriptional, translational, and microRNA-mediated control, DNA replication and maintenance of genomic stability. DHX9 has also been implicated in tumor cell maintenance and drug response. Here we report that inhibition of DHX9 expression is lethal to human cancer cell lines and murine Eµ-Myc lymphomas. Using a novel conditional shDHX9 mouse model, we demonstrate that sustained and prolonged (6 months) suppression of DHX9 does not result in any deleterious effects at the organismal level. Body weight, blood biochemistry and histology of various tissues were comparable to control mice. Global gene expression profiling revealed that, although reduction of DHX9 expression resulted in multiple transcriptome changes, these were relatively benign and did not lead to any discernible phenotype. Our results demonstrate a robust tolerance for systemic DHX9 suppression in vivo and support the targeting of DHX9 as an effective and specific chemotherapeutic approach.

A link between basic fibroblast growth factor (bFGF) and EWS/FLI-1 in Ewing's sarcoma cells.

The EWS/FLI-1 fusion gene is characteristic of most cases of Ewing's sarcoma and has been shown to be crucial for tumor transformation and cell growth. In this study we demonstrate a drastic down-regulation of the EWS/FLI-1 protein, and a growth arrest, following serum depletion of Ewing's sarcoma cells. This indicates that growth factor circuits may be involved in regulation of the fusion gene product. Of four different growth factors tested, basic fibroblast growth factor (bFGF) was found to be of particular significance. In fact, upon treatment of serum-depleted cells with bFGF, expression of the EWS/FLI-1 protein and growth of the Ewing's sarcoma cells were restored. In addition, a bFGF-neutralizing antibody, which was confirmed to inhibit FGF receptor (FGFR) phosphorylation, caused down-regulation of EWS/FLI-1. Experiments using specific cell cycle blockers (thymidine and colcemide) suggest that EWS/FLI-1 is directly linked to bFGF stimulation, and not indirectly to cell proliferation. We also demonstrated expression of FGFRs in several tumor samples of Ewing's sarcoma. Taken together, our data suggest that expression of FGFR is a common feature of Ewing's sarcoma and, in particular, that the bFGF pathway may be important for the maintenance of a malignant phenotype of Ewing's sarcoma cells through up-regulating the EWS/FLI-1 protein. Oncogene (2000) 19, 4298 - 4301