A small molecule based on the pRb2/p130 spacer domain leads to inhibition of cdk2 activity, cell cycle arrest and tumor growth reduction in vivo.

One strategy in the development of anticancer therapeutics has been to arrest malignant proliferation through inhibition of the enzymatic activity of cyclin-dependent kinases (cdks), which are key regulatory molecules of the cell cycle. Over the past few years, numerous compounds with remarkable cdk inhibitory activity have been studied in cancer therapy, although it is very difficult to point out the best cdk to target. An excellent candidate appears to be cdk2, whose alteration is a pathogenic hallmark of tumorigenesis. The small molecule described in our study showed an inhibitory effect on the kinase activity of cdk2, a significant growth arrest observed in a colony formation assay and a reduction in the size of the tumor in nude mice, thus suggesting its potential role as a promising new type of mechanism-based antitumor drug, also for the treatment of hyperproliferative disorders.

Retinoblastoma family proteins as key targets of the small DNA virus oncoproteins.

RB, the most investigated tumor suppressor gene, is the founder of the RB family of growth/tumor suppressors, which comprises also p107 (RBL1) and Rb2/p130 (RBL2). The protein products of these genes, pRb, p107 and pRb2/p130, respectively, are also known as 'pocket proteins', because they share a 'pocket' domain responsible for most of the functional interactions characterizing the activity of this family of cellular factors. The interest in these genes and proteins springs essentially from their ability to regulate negatively cell cycle processes and for their ability to slow down or abrogate neoplastic growth. The pocket domain of the RB family proteins is dramatically hampered in its functions by the interference of a number of proteins produced by the small DNA viruses. In the last two decades, the 'viral hypothesis' of cancer has received a considerable renewed impulse from the notion that small DNA viruses, such as Adenovirus, Human papillomavirus (HPV) and Polyomavirus, produce factors that can physically interact with major cellular regulators and alter their function. These viral proteins (oncoproteins) act as multifaceted molecular devices that have evolved to perform very specific tasks. Owing to these features, viral oncoproteins have been widely employed as invaluable experimental tools for the identification of several key families of regulators, particularly of the cell cycle homeostasis. Adenovirus early-region 1A (E1A) is the most widely investigated small DNA tumor virus oncoprotein, but relevant interest in human oncology is raised by the E1A-related E7 protein from transforming HPV strains and by Polyomavirus oncoproteins, particularly large and small T antigens from Simian virus 40, JC virus and BK virus.

Lonidamine, a selective inhibitor of aerobic glycolysis of murine tumor cells.

The action of Lonidamine [1-(2,4-chlorobenzyl)-1H-indazol-3-carboxylic acid] on respiration and aerobic lactate production of several murine tumor cells and normal differentiated murine cells was investigated. Lonidamine reduced the oxygen consumption in both normal and neoplastic cells. In contrast, it increased the aerobic glycolysis of normal cells but inhibited that of tumor cells. This selective action might be ascribed to the inhibition of mitochondrially bound hexokinase, which is usually absent in normal differentiated cells.

Who is the boss in the retinoblastoma family? The point of view of Rb2/p130, the little brother.

This review portrays an updated overview about the possible tumor suppressive properties of the Rb2/p130 gene, the third member of the retinoblastoma (RB) family of genes, including RB itself and p107. After a brief analysis of the established structural and functional similarities among the three genes, the main purpose is to critically analyze present evidence whether Rb2/p130 shares the role of a tumor suppressor. Taking into account the well-proven growth suppressive properties of Rb2/p130 and p107, we discuss the analysis of mutated or deleted forms of Rb2/p130 found in a number of human cancers. Finally, we take into consideration the data provided by the targeted disruption of each RB family gene, alone or in combination, in the mouse model.

Contributions of glycolysis and oxidative phosphorylation to adenosine 5'-triphosphate production in AS-30D hepatoma cells.

The AS-30D rat hepatoma cell line is characteristic of that class of rapidly growing tumors which exhibit high rates of aerobic glucose utilization and lactic acid production (Bustamante, E., Morris, H.P., and Pedersen, P.L., J. Biol. Chem., 256: 8699-8704, 1981). In this study, we have examined the coupling properties of the mitochondria in intact AS-30D hepatoma cells and the relative contributions of cytoplasmic (glycolytic) and mitochondrial compartments to total cellular ATP production in the presence of glucose and glutamine. All respiration in AS-30D cells was inhibited by inhibitors of mitochondrial electron transport, ruling out significant rates of respiration from other cellular components. Moreover, cellular respiration was found to be coupled to phosphorylation of ADP, as demonstrated by its inhibition by oligomycin and aurovertin, inhibitors of the mitochondrial ATP synthetase (F0F1-ATPase). When intact cells were supplied with glucose as the only added energy source, it was estimated that about 60% of the total cell ATP was derived from glycolysis and 40% from oxidative phosphorylation. Addition of physiological concentrations of glutamine in the presence of glucose had little effect on the relative contributions of glycolysis and oxidative phosphorylation to total cellular ATP production. In the absence of added glucose, glutamine alone could maintain the same ATP production rates by supporting mitochondrial oxidative phosphorylation. It is concluded that, in the AS-30D hepatoma cell line, glucose is the preferred energy source, with the larger portion of ATP production being supplied by glycolytic reactions. Although oxidative substrates such as glutamine can replace glucose in maintaining total cell ATP production, they do not appear to be the major fuel sources when hepatoma AS-30D cells are exposed to concentrations of substrates which occur in vivo.

Purification and characterization of a bindable form of mitochondrial bound hexokinase from the highly glycolytic AS-30D rat hepatoma cell line.

Recent studies from this laboratory have demonstrated that a form of hexokinase characteristic of rapidly growing, highly glycolytic tumor cells is bound to an outer mitochondrial membrane receptor complex containing a Mr 35,000 pore protein (D. M. Parry and P. L. Pedersen, J. Biol. Chem., 258: 10904-10912, 1983; R. A. Nakashima, et al., Biochemistry, 25: 1015-1021, 1986). In new studies reported here the specificity of this receptor complex for binding hexokinase is defined, and a purification scheme is described which leads to a homogeneous and bindable form of the tumor hexokinase. In the AS-30D hepatoma, hexokinase activity is elevated more than 100-fold relative to liver tissue. The relative increase in hexokinase activity is 8 times greater than that of any other glycolytic enzyme. Hexokinase is the only glycolytic enzyme of AS-30D cells to exhibit a mitochondrial/cytoplasmic specific activity ratio greater than 1, showing a 3.5-fold elevation in the mitochondrial fraction. Purification of hexokinase is accomplished by preferential solubilization of the mitochondrial bound enzyme with glucose-6-phosphate, followed by high-performance liquid chromatography on gel permeation and anion exchange columns. The final fraction has a specific activity of 144 units per mg of protein, with a Km for glucose of 0.13 mM and for ATP of 1.4 mM. The purified tumor enzyme migrates as a single species upon sodium dodecyl sulfate: polyacrylamide gel electrophoresis with an apparent molecular weight of 98,000. Significantly, the purified tumor enzyme retains its activity for mitochondrial binding. Additional results derived from chromatographic, polyclonal antibody, and amino acid analysis studies indicate that the predominant rat hepatoma hexokinase species is related most closely to isozymic form(s) of the enzyme commonly referred to as type II, and least related to the liver type IV isozyme (glucokinase).

Effect of lonidamine on the energy metabolism of Ehrlich ascites tumor cells.

The action of Lonidamine [1-(2,4-dichlorobenzyl)-1-H-indazol-3-carboxylic acid] on oxygen consumption and the rate of aerobic and anaerobic lactate production by Ehrlich ascites tumor cells has been investigated. The rate of oxygen consumption decreases exponentially with the increase of Lonidamine concentration, with maximal inhibition occurring at 0.40 mM Lonidamine. The rate of aerobic lactate production is inhibited to the same extent as is the oxygen consumption. However, the maximum effect is observed at 0.12 mM Lonidamine, and the decrease is linear with Lonidamine concentration. Anaerobic lactate production is more sensitive to Lonidamine, and complete inhibition can be observed by raising the concentration to 0.6 mM. The possibility that the decrease observed in lactate production was secondary to the inhibition of sodium- and potassium-containing adenosinetriphosphatase was excluded, because the drug has no effect on this enzyme. Mitochondrial adenosinetriphosphatase was not affected. Lonidamine was, however, shown to inhibit the activity of mitochondrially bound hexokinase to approximately the same extent as it inhibited aerobic glycolysis (approximately 70%). It is concluded that inhibition of the glycolysis of Ehrlich ascites tumor cells by Lonidamine results from an effect of the drug on the mitochondrially bound hexokinase.

The retinoblastoma gene product in acute myeloid leukemia: a possible involvement in promyelocytic leukemia.

The retinoblastoma susceptibility gene in leukemia and lymphoma has been investigated using different approaches involving either gene or protein analysis. In this study, a novel method, which evaluates the functional status of the retinoblastoma gene product by a binding assay to an in vitro-translated viral oncoprotein, has been applied to leukemic cells from acute myeloid leukemia patients. One hundred twenty-two cases were considered, and 42 of them were also analyzed by Western blot. Results obtained with the two methods were comparable, with the exception of few cases, where the retinoblastoma protein appeared detectable but unable to bind to the viral oncoprotein. The retinoblastoma protein has been found defective mostly in the M3 promyelocytic subtype.

Defective human retinoblastoma protein identified by lack of interaction with the E1A oncoprotein.

Inactivating mutations of the retinoblastoma susceptibility gene (Rb) are involved in the pathogenesis of hereditary and sporadic retinoblastoma. Alterations in the Rb gene have also been found in several other human tumors occurring with epidemiological incidence higher than that of retinoblastoma. Four human malignant glioma cell lines were examined for abnormalities in the retinoblastoma gene product (pRb), using a procedure based on the interaction of pRb with an in vitro-translated adenovirus E1A oncoprotein. In the CRS-A2 cell line, derived from a glioblastoma multiforme, pRb did not bind with the in vitro-translated E1A protein. Restriction analysis of the CRS-A2 Rb gene and Rb mRNA expression provided patterns that could not be distinguished from the other glioma cell lines. Further investigation revealed the presence of a truncated pRb in the CRS-A2 cell line, due to a nucleotide insertion in the coding sequence at position 2550. In addition, this truncated Rb protein was undetectable in phosphorylated form. The binding assay with the in vitro-translated E1A was also used to study other cell lines with known mutations in the Rb gene. This method, which evaluates the interaction between in vitro-translated E1A and the pRb, is proposed as a rapid screening for detecting functional alterations in the retinoblastoma protein.

Functional analysis of pRb2/p130 interaction with cyclins.

The retinoblastoma (Rb) family consists of the tumor suppressor pRb and related proteins p107 and pRb2/p130. Ectopic expression of pRb and p107 results in a growth arrest of sensitive cells in the G1 phase of the cell cycle. We demonstrated here that the growth-suppressive properties of pRb2/p130 were also specific for the G1 phase. The A-, E-, and D-type cyclins as well as transcription factor E2F1 and the E1A viral oncoprotein were able to rescue the pRb2/p130-mediated G1 growth arrest in SAOS-2 cells. The rescue with cyclins A and E correlated with their physical interaction with pRb2/p130, which surprisingly has been found to occur over all phases of the cell cycle. The phosphorylation status as well as the kinase activity associated with pRb2/p130 dramatically increased near the G1-S-phase transition. This suggests that, like the other Rb family members, pRb and p107, the phosphorylation of pRb2/p130 is controlled by the cell cycle machinery and that pRb2/p130 may indeed be another key G1-S-phase regulator.

RB1 oncosuppressor gene over-expression inhibits tumor progression and induces melanogenesis in metastatic melanoma cells.

The retinoblastoma gene (RB1) is frequently deleted or mutated in many tumor types and in all cases of retinoblastoma. Apart from its role in regulation of the cell cycle, the RB1 gene product (p110RB1) appears to be involved in control of differentiation. Malignant metastatic cells show many properties of poorly differentiated cells, and are highly invasive in vitro and in vivo. We have transfected the human RB1 cDNA in an expression vector under the control of the beta-actin promoter into B16F10 murine melanoma cells. These cells highly overexpress RB1 mRNA and the p110RB1 product, show reduced growth rate and increased melanogenesis in vitro. Vector control transfectants showed no alteration of invasiveness. The p110RB1 over-expressing cells also had a reduced capacity to migrate and invade through an artificial basement membrane, key characteristics of metastatic cells. When injected into nude mice, the p110RB1 over-expressing cells showed reduced tumor growth and reduced metastatic potential. The few metastasis observed were predominantly melanotic. These data indicate that RB1 gene expression is involved in melanoma cell differentiation and plays a role in downregulation of migration, invasion and metastatic potential of these cells.

The retinoblastoma-related Rb2/p130 gene is an effector downstream of AP-2 during neural differentiation.

Rb2/p130, a member of the Retinoblastoma family of growth and tumour suppressor genes, is extensively implicated in the control of cell cycle and differentiation. The minimal promoter region of Rb2/p130 in T98G human glioblastoma cells was identified and its analysis revealed the presence of a KER1 palindromic sequence able to bind the transcription factor AP-2, a regulatory protein that plays a crucial role in ectodermal differentiation. This KER1 site interacted in vitro with AP-2, and AP-2 overexpression increased Rb2/p130 transcription and translation. We also found that rat PC12 pheochromocytoma cells, when induced to differentiate by NGF, displayed an increase of AP-2 protein levels and of Rb2/p130 transcription and protein levels. AP-2-transfected PC12 cells displayed enhanced transcription and translation of Rb2/p130 and of the cdk inhibitor p21(WAF1/CIP1), a gene known to be under the control of AP-2, but unable by itself to elicit PC12 differentiation. Overexpression of either AP-2 or Rb2/p130 elicited per se cell differentiation in the absence of NGF, while coexpression of AP-2B, a negative regulator of AP-2 transcriptional activity, inhibited only AP-2-induced differentiation. Altogether, these results indicate that Rb2/p130 is a critical effector of AP-2 in sustaining ectodermal differentiation.

A serine 37 mutation associated with two missense mutations at highly conserved regions of p53 affect pro-apoptotic genes expression in a T-lymphoblastoid drug resistant cell line.

The p53 protein accumulates rapidly through post-transcriptional mechanisms following cellular exposure to DNA damaging agents and is also activated as a transcription factor leading to growth arrest or apoptosis. Phosphorylation of p53 occurs after DNA damage thereby modulating its activity and impeding the interaction of p53 with its negative regulator oncogene Mdm2. The serines 15 and 37 present in the amino terminal region of p53 are phosphorylated by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. In order to verify if specific p53 mutations occur in the multi-drug resistance phenotype, we analysed the p53 gene in two T-lymphoblastoid cell lines, CCRF-CEM and its multi-drug-resistant clone CCRF-CEM VLB100, selected for resistance to vinblastine sulfate and cross-resistant to other cytotoxic drugs. Both cell lines showed two heterozygous mutations in the DNA binding domain at codons 175 and 248. The multi-drug resistant cell line, CCRF-CEM VLB100, showed an additional mutation that involves the serine 37 whose phosphorylation is important to modulate the protein activity in response to DNA damage. The effects of these mutations on p53 transactivation capacity were evaluated. The activity of p53 on pro-apoptotic genes expression in response to DNA damage induced by (-irradiation, was affected in the vinblastine (VLB) resistant cell line but not in CCRF-CEM sensitive cell line resulting in a much reduced apoptotic cell death of the multi-drug resistant cells.

Interplay between the antimetastatic nm23 and the retinoblastoma-related Rb2/p130 genes in promoting neuronal differentiation of PC12 cells.

Increasing evidence indicates that the nm23 genes, initially documented as suppressors of metastasis progression, are involved in normal development and differentiation. We have shown previously that the murine nm23 gene enhances pheochromocytoma PC12 cells responsiveness to NGF by accelerating cell growth arrest and neurite outgrowth. The present study was aimed at elucidating the mechanisms by which nm23 controls cell proliferation and promotes neuronal differentiation. We demonstrated that nm23 modulates the expression of the Rb2/p130 gene, a negative regulator of cell cycle progression also implicated in the maintenance of the differentiated state. Furthermore, we showed that nm23-H1 mutants, defective in inhibiting the invasive phenotype, downregulate Rb2/p130 expression and inhibit NGF-induced PC12 cell differentiation. In synthesis, our results provide first evidence of interplay between the nm23 and the Rb2/p130 genes in driving PC12 cells neuronal differentiation and suggest that the antimetastatic and the differentiative nm23 functions can have similar features.

The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation.

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.

Effect of bcr-abl oligodeoxynucleotides on the clonogenic growth of chronic myelogenous leukaemia cells.

We studied the effect of phosphorothioate oligodeoxynucleotides ([S]ODNs) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukaemia (CML). Experiments included the evaluation of the anti-leukaemic effect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34+ cells, evaluation of incubation time and correlation of colony growth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34+ cell populations and the cross-hybridization of 26- and 16-mer [S]ODNs with the complementary sequences were evaluated. After incubation for 120 h with 26-mer antisense [S]ODNs on mononuclear cells, overall mean colony recovery was 41.9% of the untreated control samples; in particular, a significant reduction in colony formation was observed in 22 of the 35 cases tested. The effect of 26-mer ODNs on CD34+ cells was comparable to that observed on mononuclear cells in terms of colony inhibition; however, a higher proportion of cases showed a significant inhibition of colony formation. In comparison with the 26-mer antisense [S]ODNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was less evident on mononuclear cells and comparable on CD34+ cells; however, a more specific effect was evident on both target cells. Hybridization experiments confirmed a partial cross-reactivity when the 26-mer ODNs were hybridized with their complementary sequence; this did not occur when 16-mer ODNs were similarly tested. Experiments aimed at evaluating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to that measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs from day 1 to day 5. The accumulation of [S]ODNs correlated with a marked down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The down-regulation of p210(bcr-abl) levels following treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expression. These studies confirm that, under optimal conditions of target cell culture and ODN size, antisense [S]ODNs complementary to the bcr-abl junction have specific anti-leukaemic effects.

Pattern of expression of cyclin T1 in human tissues.

Cyclin T1 was recently identified, together with cdk9 (previously named PITALRE), as part of the TAK multiprotein complex, a co-factor targeted by the human immunodeficiency virus Type 1 (HIV-1) protein named Tat, suggesting a role for this complex in transcription elongation. Although studies on mRNA and protein expression have shown that cyclin T1 is ubiquitous in adult human tissues, no data have yet been reported regarding the expression of this protein in different cell lineages. Using a polyclonal antiserum raised against cyclin T1, we investigated the pattern of expression of this protein in adult human tissues by immunohistochemistry. Cyclin T1 was expressed ubiquitously, although different levels of expression were found in various organs. Some specialized tissues, such as blood, lymphoid tissues, and cells of connective tissue origin, showed high cyclin T1 expression. These specific expression patterns are only partially justified by some well-known specialized functions of cyclin T1 in certain cell types, such as its involvement in peripheral blood lymphocytes and monocyte differentiation. The high expression level found in other tissues suggests new possible roles for cyclin T1 in cell types other than those of lymphoid tissue.

Cyclin T2a gene maps on human chromosome 2q21.

Cyclin T2a was recently identified as one of the regulatory subunits of the cdk-cyclin complex P-TEFb, the most studied positive factor in the regulation of transcription elongation. By fluorescent in situ hybridization (FISH), the gene codifying for cyclin T2a has been mapped on human chromosome 2q21. This locus also has been linked to different forms of myopathy. By use of a new specific antiserum raised against cyclin T2a, the immunohistochemical pattern of expression of cyclin T2a in human tissues has been examined and compared to that of cyclin T1, described in the previous report. The observation that immunohistochemical expression of cyclin T2a was high in skeletal muscle cells, whereas it was undetectable in two cases of centronuclear myopathy, together with its chromosomal location, suggests an involvement of the cdk9-cyclin T2a complex in this disease.

Inhibition of protein synthesis in neoplastic cells by rhein.

The action of rhein, 4,5-dihydroxyanthraquinone-2-carboxylic acid, on protein synthesis of neoplastic cells has been investigated. Rhein decreases amino acid incorporation in all cells tested. The inhibition of incorporation of labeled precursors into acid-insoluble material cannot be ascribed to an impairment of amino acid uptake, which is unaffected by the drug. Tests on cell-free system showed that rhein does not inhibit the TMV-mRNA directed in vitro protein synthesis, thus indicating that the protein machinery per se is not affected. The inhibition of protein brought about by the drug must be ascribed to an effect on the energy-yielding processes with a remarkable decrease in ATP content. The mechanism is similar to that of other metabolic inhibitors, but rhein, for its capability to inhibit both respiration and glycolysis, is effective at much lower concentrations.

Enhancement of doxorubicin content by the antitumor drug lonidamine in resistant Ehrlich ascites tumor cells through modulation of energy metabolism.

The effect of the antitumor drug lonidamine (LND) on respiration, aerobic glycolysis, adenylate pool, doxorubicin (DOX) uptake, and efflux in DOX-resistant and DOX-sensitive Ehrlich tumor cells was investigated. The results may be summarized as follows: 1) In both types of cells, LND inhibited both respiration and glycolysis in a dose-dependent manner and lowered the ATP concentration. The effect was more marked in cells incubated in glucose-free medium; 2) LND raised, to a remarkable extent, the intracellular content of DOX in resistant and sensitive cells respiring on endogenous substrates because of reduced ATP availability, whereas in glucose-supplemented medium, where both respiration and glycolysis contributed to ATP synthesis, the increase was lower; and 3) when LND was added to DOX-loaded cells, it failed to significantly inhibit DOX efflux because of time-dependent phenomena. These findings indicated that LND, a drug currently employed in tumor therapy, might also be useful in reducing or overcoming multidrug resistance (MDR) of those cells with a reduced ability to accumulate and retain antitumor drugs.