Induction of plasminogen activator by alkylating agents in a repair defective human glioblastoma cell strain.

Alkylating agents, mechlorethamine and N-methyl-N'-nitro-N-nitrosoguanidine, induce the production of plasminogen activator in U-87MG cells, an alkylation DNA repair deficient (Mer-) human glioblastoma strain. Enzyme induction was not observed, however, in U-178MG and SH-101 cells, alkylation repair proficient (Mer+) glioblastoma strains, or in HeLa cells, which reactivated and supported well the growth of alkylation damaged adenovirus 3. In the alkylation repair defective U-87MG strain, enhanced production of plasminogen activator occurred in a narrow concentration range of treatment with either alkylating agent, causing a 20 to 50% inhibition of [3H]thymidine incorporation. Maximum plasminogen activator induction was observed between 32 and 48 h after alkylation treatment and the levels of enzyme produced were 5 to 10 times those of untreated control levels. This alkylation dependent enzyme induction required protein synthesis for it did not occur in the presence of cycloheximide. It was hence concluded that plasminogen activator induction in alkylation repair deficient human cells is caused by unrepaired DNA damage and that it may represent an eukaryotic SOS-like function. In addition, plasminogen activator induction may be useful as a sensitive assay for the identification of alkylation repair defective human tumors for which the susceptibility to alkylation chemotherapy should be expected to increase.

Production of plasminogen activator by synchronized normal and Rous sarcoma virus-transformed chicken fibroblasts in culture.

We studied intracellular activity of the plasminogen activator within the cell cycle of chemically synchronized normal and RSV-transformed chick fibroblasts in culture. Consideration has also been given to the relationship between the plasminogen activator activity and cycles of DNA synthesis or mitosis in cycling fibroblasts after viral infection. The plasminogen activator activity of the cell lysates was assayed on [125I]fibrin-coated Petri dishes and was expressed as the radioactivity released from the plates. Normal fibroblasts produced detectable levels of plasminogen activator in the S-phase and late G2-phase or mitosis of the cell cycle. In contrast, RSV-transformed cells produced high levels of this activator throughout the entire cell cycle although this activity fluctuated and reached a maximum in the G2-M periods. We also found that the level of plasminogen activator activity in the transformed fibroblasts is influenced by the cycles of DNA synthesis and that cell division is required for the appearance of plasminogen activator activity in the 'de novo' virus-infected cultures.

Inhibition of viral RNA synthesis by 1-methyl-1-nitrosourea.

The effect of 1-methyl-1-nitrosourea on the synthesis of viral RNA of Poliovirus in KB-cells has been examined. It was found that 1-methyl-1-nitrosourea at concentration of 400 micrograms/ml blocked the synthesis of viral RNA in Poliovirus-infected KB-cells irrespective of whether it was added to culture medium immediately after virus adsorption or 2 h later. Furthermore, the inhibitory effect of 1-methyl-1-nitrosourea on the growth of Poliovirus is comparable to that of Herpes simplex virus grown in KB-cells. These findings suggest that 1-methyl-1-nitrosourea does not discriminate between these two types of viruses and moreover that the viral RNA of Poliovirus could also be the target for the action of the drug.

Dimethyl-10,12-benz(a)acridine; evidence for differential effects on the synthesis of RNA of mammalian or avian fibroblasts and some RNA viruses.

We have studied the differential effect of dimethyl-10,12-benz(a)acridine (DBMAcr) on the synthesis of RNA of chicken or mouse fibroblasts in culture and that of some RNA-containing viruses such as Rous sarcoma virus and Mengovirus. DMBAcr at low concentrations blocks the cell multiplication of both normal and Rous sarcoma virus-transformed chicken fibroblasts in culture; it affects transformed cells more than normal ones. The cell growth inhibiting effect of DMBAcr is reversible after short periods of incubation. DMBAcr depresses the synthesis of cellular DNA and RNA in parallel. Concurrently the synthesis of protein proceedes at a relatively high rate in DMBAcr-treated cultures. Its inhibitory effect on cellular RNA synthesis is mostly due to a block in the formation of 28 S and 18 S ribosomal RNA species; in contrast, the synthesis of 45 S ribosomal RNA precursor is proceeding at almost control rate. Also, the synthesis of heterogeneous nuclear RNA is not blocked by DMBAcr. The production of Rous sarcoma virus in transformed fibroblasts is not affected by DMBAcr. Since this is correlated with persisting high rates of protein and heterogenous nuclear RNA synthesis, the effects of DMBAcr suggest that the synthesis of Rous sarcoma virus-RNA shares the specificity of messenger and heterogeneous nuclear RNA. DMBAcr inhibits the synthesis of viral RNA of Mengovirus under conditions where the synthesis of total cellular RNA is not appreciably depressed, suggesting its differential effect on the DNA-directed and the RNA-directed RNA synthesis.

Conformation and structure of 2-amino-8-(beta-D-ribofuranosyl)imidazo [1,2-a]-s-triazin-4-one(5-aza-7-deaza guanosine), a potent antiviral nucleoside.

The crystal and molecular structure of one imidazo[1,2-a]-s-triazine nucleoside and its antiviral activity are described. The crystal structure of 2-amino-8-(beta-D-ribofuranosyl)imidazo[1,2-a]-s-triazin-4-one monohydroate (C10H13N5O5.H2O) was solved by X-ray counter data. The compound crystallizes in the monoclinic space group P21 with cell dimensions a = 7.353 (1), b = 6.465 (1), c = 13.701 (1) A, B = 104.64 (1) degrees. The structure was solved by direct methods and refined by full matrix least-squares technique to a final value of the conventional R-factor of 0.049 using 1998 observed intensities. The orientation of the base relative to the sugar ring defined in terms of rotating about the C(1')-N(8) glycosyl bond is anti (47.8 degrees). The ribose moiety exhibits C(2')-endo, E conformation. The conformation around C(4')-C(5') is gauche-. Molecular packing is dominated by hydrogen bonds. Base stacking occurs long the b axis. 5-Aza-7-deazaguanosine has shown a marked antiviral activity in vitro against herpes simplex virus despite the fact that N(3) is effective as the hydrogen acceptor only.

Structure and function of microplasminogen: I. Methionine shuffling, chemical proteolysis, and proenzyme activation.

We have cloned and expressed microplasminogen (mPlg), consisting of the N-terminal undecapeptide of human glu-Plg spliced to its proenzyme domain. This truncated (approximately 28 kDa) proenzyme retained the distinctive catalytic activities of the larger parent. Replacement of M residues followed by M shuffling permitted subsequent scission by site-directed chemical proteolysis (in CNBr/formic acid) without impairing any of the protein's characteristic properties. Activation of chymotrypsinogen-related zymogens occurs by limited proteolysis; the newly liberated, highly conserved N-terminus (VVGG) forms a salt bridge with an aspartyl residue immediately upstream of the active site serine. The role of both of these elements in mPlg activation was probed using protein engineering and site-directed proteolysis to alter the length and amino acid composition of the N-terminus, and to replace the aspartate. All modifications affected both Km and Kcat. The results identify some structural parameters of the N-terminus required for proenzyme activation.

Sudden senescence syndrome plays a major role in cell culture proliferation.

Normal human cells of various types have a finite and predictable proliferative potential in vitro. This limited life span is due to a gradually increasing fraction of senescent cells that appear in the culture in a sudden and stochastic fashion due to a phenomenon referred to as sudden senescence syndrome (SSS). Because nondividing cells increasingly accumulate in the culture, dividing cells have to compensate for nondividers in order to accomplish additional population doubling (PD). Thus, individual dividing cells undergo more divisions, called cell generations (CG), than the number of PDs. Based on integrated experimental data, we calculated maximum CG for normal human diploid fibroblasts (HDF). It appears that for a HDF culture that undergoes 65 PD, the calculated final CG is at least 126. Based on the obtained value for CG we calculated the total size of the culture, both with and without effect of SSS. If no SSS takes place and cells divide by geometrical progression, the culture will grow up to 2(126) or 10(38) cells. By constantly eliminating cells from further divisions, causing cell loss (CL), SSS reduces the total size of the culture at every point during its proliferation. The calculated value for CL is enormous, so that the culture of 10(38) cells is reduced to only 10(19) cells, thus as little as 10(-17)% of its size! Accordingly, by preventing virtually every cell in the culture from reaching its original maximum doubling capacity, SSS appears to be the most important mechanism that influences cell culture proliferation.

The effects of 1-methyl-1-nitrosourea on the survival pattern of cycling and noncycling HeLa cells.

The biological behavior of HeLa cells exposed to 1-methyl-1-nitrosourea was examined by determining the survival fraction in asynchronous and synchronous cultures. Asynchronous cell population exposed to 1-methyl-1-nitrosourea for 1 h exhibited a shoulder type survival curve, indicating that damage must be accumulated before the lethal effect occurs. A fraction of 25% of cells survives the concentration of 100 micrograms/ml. The duration of treatment with the drug did not have a significant effect on the cell survival. The experiments with synchronized cells showed that MNU exhibits the killing in all phases of the cell age, but the most sensitive are these in S phase. However, they are still six times more resistant at the same concentration than the culture in plateau phase. At the concentration of 100 micrograms/ml nondividing plateau cells are about 35 times more sensitive than exponentially growing cells. We can conclude that MNU acts as the most acting killing agents on the cells which are in nondividing plateau phase.

Quality control program of steroid receptor assays: an international study.

Lyophilized calf uterine cytosol standards were prepared for quality control of estrogen receptor (ER) determination, and lyophilized cytosols and tissue powders were used for quality control of progesterone receptor (PR) analysis. Two series of four samples were analyzed either for ER or PR contents, twice within one month, by 7 laboratories in 5 countries. Coefficient of variation (CV) of the between-laboratory averages assayed in a single run of ER-positive (ER+) and PR-positive (PR+) standards varied from 29.6 to 61.8% and from 32.4 to 76.2%, respectively. All laboratories, with the exception of a single value, could recognize samples of low, medium, an high ER level, as well as a negative sample. Most laboratories evaluated properly also the level of PR samples. The average between-laboratory CV values of protein determination in the relevant standards were 23%.

Estrogen and progesterone receptors as prognostic factors in breast cancer.

The relation between estrogen receptors (ER) and/or progesterone receptors (PgR) and some clinical factors such as tumor size, axillary node involvement, histological tumor grade, and disease-free interval (DFI) in 500 patients with operable (TNM stage I-III) breast cancer was studied. ER-positive (ER+) tumors were commoner in older patients, whereas PgR-positive (PgR+) tumors were similarly distributed within the age groups. The concentration of ER+ protein also increased with age in contrast to PgR+ protein concentration. However, receptor status was not associated with menopausal status independently of age. Axillary node involvement influenced neither ER nor PgR status, but there was a statistically significant relation between tumor size and positivity of ER or PgR. There was no association between histologic tumor grade and either steroid receptor phenotype. DFI was longer in patients with ER+ than those with ER- tumors, independently of axillary nodal status. The positivity of PgR in patients with ER+ tumors contributed to an even longer DFI, suggesting that the combination of ER/PgR is a better indicator of DFI than ER or PgR alone.

Induction of plasminogen activator by N-methyl-N'-nitro-N-nitrosoguanidine in mer+ and mer- human tumour cell strains.

Plasminogen activator (PA) synthesis in alkylation DNA repair deficient (mer-) and proficient (mer+) human tumour cell strains exposed to an alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), has been studied. MNNG enhanced the production of PA in mer- cell strains (U87MG, A1235, A1336, A172), but not in mer+ strains (TE85, HT29, U178MG, A288), which reactivated and supported well the growth of alkylation damaged adenovirus 3. Several mer+ strains (A549 and A2182), which are highly susceptible to killing by MNNG, produced moderately elevated enzyme levels after alkylating treatment. In the alkylation repair defective strains, enhanced production of both intra and extracellular PA occurred with 2-10 microM MNNG causing a 20-40% inhibition of [3H]thymidine incorporation. Maximum PA induction was observed 30-48 h after alkylation treatment and the levels of enzyme produced were 5-10 times as high as those of untreated control levels. As shown by electrophoretic analysis, MNNG enhances in mer- cells the synthesis of 40,000-50,000 Dalton of human urokinase type PA which is also present in lower amounts in untreated cells. This alkylation induced PA production by mer- cells required RNA and protein synthesis because it did not occur in the presence of actinomycin D or cycloheximide. PA induction by MNNG occurred throughout the cell cycle of synchronized mer- cells indicating that blockage of DNA synthesis is not responsible for enzyme induction and that it may result from DNA transcription on a damaged template. It was thus concluded that PA induction is causally associated with deficient DNA repair, which makes it useful as a sensitive assay for identification of alkylation repair deficient cell strains.

Selective irreversible inactivation of replicating mengovirus by nucleoside analogues: a new form of viral interference.

We describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (BrTu) and tubercidin (Tu). At a concentration of 5 microgram/ml, BrTu reversibly blocked the synthesis of cellular mRNA and rRNA but did not inhibit either mengovirus RNA synthesis or multiplication. BrTu is a potent inhibitor of adenosine kinase, and low concentrations of BrTu (e.g., 0.5 microgram/ml), which did not by themselves inhibit cell growth, blocked phosphorylation of Tu and thus protected uninfected cells against irreversible cytotoxicity resulting from Tu incorporation into nucleic acids. In contrast, in mengovirus-infected cells, BrTu did not completely inhibit Tu incorporation into mengovirus RNA, allowing the formation of Tu-containing functionally defective polynucleotides that aborted the virus development cycle. This increased incorporation of Tu coupled to mengovirus infection could be attributed either to a reduction in the inhibitory action of BrTu and/or its nucleotide derivatives at the level of nucleoside and nucleotide kinases and/or, perhaps, to an effect upon the nucleoside transport system. The virus life cycle in nucleoside-treated cells progressed to the point of synthesis of negative strands and probably to the production of a few defective new positive strands. Irreversible virus growth arrest was achieved if the nucleoside mixture of BrTu (0.5 to 10 microgram/ml) and Tu (1 to 20 microgram/ml) was added no later than 30 min after virus infection and maintained for periods of 2 to 8 h. The cultures thus "cured" of mengovirus infection could be maintained and transferred for several weeks, during which they neither produced detectable virus nor showed a visible cytopathic effect; however, the infected and cured cells themselves, while metabolically viable, were permanently impaired in RNA synthesis and unable to divide. Although completely resistant to superinfecting picornaviruses, they retained the ability to support the growth of several other viruses (vaccinia virus, reovirus, and vesicular stomatitis virus), showing that cured cells had, in general, retained the metabolic and structural machinery needed for virus production. The resistance of cured cells to superinfection with picornaviruses seemed attributable neither to interferon action nor to destruction or blockade of virus receptors but more likely to the consumption of some host factor(s) involved in the expression of early viral functions during the original infection.

Fibrinolysis associated with human neoplasia: production of plasminogen activator by human tumours.

The plasminogen-dependent fibrinolytic activity of several human tumours has been studied. The following neoplastic tissues, which were obtained from cancer patients by either surgery or biopsies, were examined: cervical, mammary, prostatic, ovarian, lung carcinomas, melanoma, basalioma. Fibrinolysis was measured in lysates either of tumour tissue specimens or of respective cell cultures. This measurement was based on release of radioactivity from 125I-fibrin-coated Petri dishes. All examined tumours showed elevated levels of plasminogen-dependent fibrinolytic activity. However, normal control tissues had little or no activity. These results indicate that the fibrinolysis might be useful in differentiating between malignant and normal tissues.

Synthesis and antiviral activities of 2-substituted azino-3-(D-arabinofuranosyl)-5-carboxymethylene-thiazolidine-4-ones.

2-(1-Isopropylidene)azino-3-beta and 3-alpha-D-arabinofuranosyl-5-methoxycarbonylmethylenethiazolidine-4-ones (III) and (IV) have been synthesized by condensation of benzylated alpha-chloro-D-arabinofuranose (V) with o-(trimethylsilyl)-2-(1-isopropilidene)azino-5-methoxycarbonylmethylen- thiazolidine-4-one (VI) in the presence of SnCl4 or molecular sieves. Condensation of benzoylated alpha, beta-bromo-D-arabinose (VII) with (VI) led exclusively to the alpha anomer (VIII). 2-(1-Isopropylidene)azino-3-alpha-D-arabinofuranosyl-5-carbamoylmethylene- thiazolidine-4-one (IX) has been synthesized from (IV) (R, R1, R2 = CH3). 1H-N.M.R. and 13C-N.M.R. data confirmed the structures of (III) and (IV) (R, R1, R2 = CH3 and R, R1 = CH3, R2 = Ph). Antiviral activities were tested on monolayer cultures of KB-cells. Whereas previously tested beta-D-ribofuranosyl derivatives had moderate activity, alpha and/or beta-D-arabinofuranosyl derivatives are totally inactive against herpes simplex and poliovirus.