The mixed lineage kinase DLK is oligomerized by tissue transglutaminase during apoptosis.

Current evidence suggests that the mixed lineage kinase family member dual leucine zipper-bearing kinase (DLK) might play a significant role in the regulation of cell growth and differentiation, particularly during the process of tissue remodeling. To further explore this working model, we have investigated the regulation of host and recombinant DLK in NIH3T3 and COS-1 cells undergoing apoptosis. Using calphostin C, a potent and selective inhibitor of protein kinase C and a recognized apoptosis inducer for various cell types, we demonstrate, by immunoblot analysis, that DLK protein levels are rapidly and dramatically down-regulated during the early phases of apoptosis. Down-regulation in calphostin C-treated cells was also accompanied by the appearance of SDS- and mercaptoethanol-resistant high molecular weight DLK immunoreactive oligomers. Experiments aimed at elucidating the mechanism(s) underlying DLK oligomerization revealed that the tissue transglutaminase (tTG) inhibitor monodansylcadaverine antagonized the effects of calphostin C almost completely, thereby suggesting the involvement of a tTG-catalyzed reaction as the root cause of DLK down-regulation and accumulation as high molecular weight species. In support of this notion, we also show that DLK can serve as a substrate for tTG-dependent cross-linking in vitro and that this covalent post-translational modification leads to the functional inactivation of DLK. Taken together, these observations suggest that transglutamination and oligomerization may constitute a relevant physiological mechanism for the regulation of DLK activity.

p53 status, cellular recovery and cell cycle arrest as prognosticators of in vitro radiosensitivity in human pancreatic adenocarcinoma cell lines.

PURPOSE: To investigate the factors contributing to the in vitro radiosensitivity of four human pancreatic adenocarcinoma cell lines differing in p53 status, and the basis for the lack of post-irradiation G1 arrest in the two cell lines that have retained a wild-type p53 allele. MATERIALS AND METHODS: Cells were X-irradiated and the parameters related to radiosensitivity, as well as the modulation of gene products linked to regulation of cell cycle transit (p53, p21/WAF1/CIP1, pRb) or DNA replication and repair (DNA topoisomerase I and II), were determined. RESULTS: Both cell lines expressing either mutant (mt) R248W or R273H p53 proteins were more radioresistant. All the cell lines arrested in G2. None of the cell lines arrested in G1 and this was linked to the inability to upregulate p21/WAF1/CIP1. There were no correlations between p53 status and the magnitude or time of maximum G2 arrest. However, there was a negative correlation between a protracted arrest in G2 and the ability to recover from potentially lethal damage (PLD). CONCLUSIONS: Variation in radiosensitivity is related to p53 status, but the survival advantage conferred by having mutant p53 status is not readily explained neither by recovery from PLD nor by cell cycle arrest kinetics. There is no p53-independent pathway for the recruitment of p21 in these cell lines following irradiation.

Inhibitory and stimulatory effects of somatostatin on two human pancreatic cancer cell lines: a primary role for tyrosine phosphatase SHP-1.

Somatostatin (SS-14) and its structural analogue SMS 201-995 (SMS) are recognized as physiological inhibitors of multiple organs and tissue functions through specific membrane receptors (sst1-sst5). The effects of SS-14 and SMS in the growth control of the pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were investigated to identify and clarify the intracellular events involved. In PANC-1 cells, SS-14 and SMS caused inhibition of their basal growth, and that stimulated by epidermal growth factor, with a maximal effect at 0.1-1 microM. To understand the inhibitory mechanisms, we investigated the effects of SS-14 and SMS on phosphotyrosine phosphatase (PTPase) activity and, more specifically, that of tyrosine phosphatase SHP-1 (PTP1C). SS-14 and SMS caused significant increases in total cellular PTPase activity, and particularly SHP-1, with maximal activation within 1 min. Inhibition of membrane tyrosine kinase and p42 MAP kinase activities was also observed, in response to SS-14 and SMS. In MIA PaCa-2 cells, SS-14 and SMS were associated with a positive growth response at 1-10 nM, after 4 days of culture in serum-free medium. Total cellular PTPase activity was slightly increased, but SHP-1 activity could not be detected; its absence in this cell line was confirmed by Western blot. Membrane tyrosine kinase activities were significantly increased by SS-14 and SMS at concentrations needed for maximal growth. p44/p42, which are constitutively active in this cell line, and p38 activities were not affected by somatostatin. In conclusion, somatostatin can exert different effects on human pancreatic cancer cell growth, depending upon the presence or absence of SHP-1. This enzyme can play a key role in the control of cell proliferation, and its cellular presence may determine the therapeutic potential of somatostatin in the control of cancer cell growth.

DNA topoisomerase I content of a pair of human melanoma cell lines with very different radiosensitivities correlates with their in vitro sensitivities to camptothecin.

In this study, we set out to determine if the differential sensitivities to CPT between a radioresistant (Sk-Mel-3) and a radiosensitive (HT-144) human melanoma cell line, and also between cultures with a different growth phase in each cell line, were related to endogenous differences in cellular transport of CPT or to DNA topo I catalytic activities and content. Cultures of HT-144 and Sk-Mel-3 cells in both the exponential, or plateau (i.e. confluent), phase of growth were compared. Cellular response to CPT was determined by clonogenic survival assay. Drug accumulation and efflux were determined using [3H]CPT. Topo I catalytic activity was determined from the ability of nuclear extracts prepared from the cells to relax supercoiled DNA plasmid. Nuclear extracts of the cells were also used to determine topo I content by western blotting. The significantly enhanced sensitivity of exponential-phase, relative to plateau-phase, cultures of both cell lines was related to an enhanced accumulation of [3H]CPT in one (i.e. HT-144), but not the other, cell line. Thus the higher sensitivity of exponential-phase cultures of HT-144 relative to those of Sk-Mel-3 can at least be partially accounted for on the basis of a relatively higher accumulation. However, a higher accumulation was not the reason why plateau-phase cultures of HT-144 were relatively more sensitive than those of Sk-Mel-3. Although there were no significant differences (at the p < 0.05 level) between the endogenous catalytic activities of topo I extracted from exponential- and plateau phase-cultures of both these cell lines, there was a trend for HT-144 cells to show higher endogenous topo I catalytic activities compared to Sk-Mel-3 cells. In contrast, topo I content was higher in exponential- relative to plateau phase-cultures of both cell lines, and in HT-144 relative to Sk-Mel-3 when cultures in a similar growth phase were compared. The relative differences in sensitivity to CPT observed in vitro between these two cell lines, and also between exponential- and plateau phase-cultures of each cell line, correlates best with topo I content rather than topo I catalytic activity or [3H]CPT transport.

Interlaboratory comparison of autoradiographic DNA profiling measurements: precision and concordance.

Knowledge of the expected uncertainty in restriction fragment length polymorphism (RFLP) measurements is required for confident exchange of such data among different laboratories. The total measurement uncertainty among all Technical Working Group for DNA Analysis Methods laboratories has previously been characterized and found to be acceptably small. Casework cell line control measurements provided by six Royal Canadian Mounted Police (RCMP) and 30 U.S. commercial, local, state, and Federal forensic laboratories enable quantitative determination of the within-laboratory precision and among-laboratory concordance components of measurement uncertainty typical of both sets of laboratories. Measurement precision is the same in the two countries for DNA fragments of size 1000 base pairs (bp) to 10,000 bp. However, the measurement concordance among the RCMP laboratories is clearly superior to that within the U.S. forensic community. This result is attributable to the use of a single analytical protocol in all RCMP laboratories. Concordance among U.S. laboratories cannot be improved through simple mathematical adjustments. Community-wide efforts focused on improved concordance may be the most efficient mechanism for further reduction of among-laboratory RFLP measurement uncertainty, should the resources required to fully evaluate potential cross-jurisdictional matches become burdensome as the number of RFLP profiles on record increases.

Ensuring recovery of intact RNA from rat pancreas.

The isolation of intact RNA from rat pancreas is compromised by autolysis and by the presence of endogenous ribonucleases. In order to ameliorate recovery we systematically investigated available RNA extraction methods and paid particular attention to the influence of frozen storage and ribonuclease inhibition strategies on overall yield and quality of RNA. Modifications to the basic procedure of Chomczynski and Sacchi (1987) are described which allow, reproducibly, to obtain rat pancreatic RNA suitable for Northern blot hybridization, RT-PCR, and differential display analysis.

CYP2C19 genotyping and associated mephenytoin hydroxylation polymorphism in a Canadian Inuit population.

The CYP2C19-associated oxidation polymorphism of mephenytoin was investigated in an Inuit population living in the high Arctic of Canada. Results were obtained for 152 subjects, of whom 90 were unrelated to first degree relatives. Phenotyping was based on the capillary gas chromatographic determination of the S/R enantiomeric ratio in overnight urine collected after a dose of 100 mg (R,S)-mephenytoin. The phenotype was confirmed by determining the S/R enantiomeric ratio after acid treatment of urine samples, and for some subjects, by determining urinary recovery of 4'-hydroxymephenytoin using capillary electrophoresis analysis. DNA was analysed for the m1 and m2 mutations of CYP2C19. Three of 152 subjects (2.0%; 95% confidence limits: 0.0-4.2%) were phenotypically classified as poor metabolizers (PMs). Genotype analysis characterized three individuals as homozygous, and 28 individuals as heterozygous for the m1 mutation, the remaining individuals being homozygous for the wild-type allele. The genotype of the three PMs was concordant with that of the phenotype. DNA fingerprinting confirmed that these three individuals were genetically unrelated. The allele frequency of the CYP2C19m1 mutation, determined in unrelated subjects, was 0.12 (95% confidence limits: 0.07-0.17). CYP2C19m2 was not detected in this population. Thus, the Canadian Inuit resemble Caucasian rather than Asian populations in both the incidence of PM phenotype and the molecular basis of the polymorphism.

Characterization of human lymphoid cell lines GM9947 and GM9948 as intra- and interlaboratory reference standards for DNA typing.

The incorporation of reference DNA is crucial to the validation of any DNA typing protocol. Currently, reference DNA standards are restricted to molecular size DNA ladders and/or tumor cell line DNA. Either of these, however, presents some limitations. We have rigorously characterized two Epstein-Barr virus (EBV)-immortalized human lymphoid cell lines--GM9947 (female) and GM9948 (male)--to determine their suitability as alternative in-line standards for three widely employed allele profiling strategies. Twenty-one highly polymorphic VNTR-based allelic systems (7 RFLPs, 2 AmpFLPs, and 12 STRs) distributed over 12 chromosomes were scrutinized along with 3 gender-based discriminatory systems. The genetic stability of each locus was confirmed over a period of 225 in vitro population doublings. Allele size estimates and degree of informativeness for each of the 21 VNTR systems were compiled. The reproducibility of allele scoring by traditional RFLP analyses, using both cell lines as reference standards, was also verified by an interlaboratory validation study involving 13 analysts from two geographically distinct forensic laboratories. Taken together, our data indicate that GM9947 and GM9948 genomic DNAs could be adopted as reliable reference standards for DNA typing.

Abnormal pattern of post-gamma-ray DNA replication in radioresistant fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome.

Non-malignant dermal fibroblast strains, cultured from affected members of a Li-Fraumeni syndrome (LFS) family with diverse neoplasms associated with radiation exposure, display a unique increased resistance to the lethal effects of gamma-radiation. In the studies reported here, this radioresistance (RR) trait has been found to correlate strongly with an abnormal pattern of post-gamma-ray DNA replicative synthesis, as monitored by radiolabelled thymidine incorporation and S-phase cell autoradiography. In particular, the time interval between the gamma-ray-induced shutdown of DNA synthesis and its subsequent recovery was greater in all four RR strains examined and the post-recovery replication rate was much higher and was maintained longer than in normal and spousal controls. Alkaline sucrose sedimentation profiles of pulse-labelled cellular DNA indicated that the unusual pattern of DNA replication in irradiated RR strains may be ascribed to anomalies in both replicon initiation and DNA chain elongation processes. Moreover, the RR strain which had previously displayed the highest post-gamma-ray clonogenic survival was found to harbour a somatic (codon 234) mutation (presumably acquired during culture in vitro) in the same conserved region of the p53 tumour-suppressor gene as the germline (codon 245) mutation in the remaining three RR strains from other family members, thus coupling the RR phenotype and abnormal post-gamma-ray DNA synthesis pattern with faulty p53 expression. Significantly, these two aberrant radioresponse end points, along with documented anomalies in c-myc and c-raf-1 proto-oncogenes, are unprecedented among other LFS families carrying p53 germline mutations. We thus speculate that this peculiar cancer-prone family may possess in its germ line a second, as yet unidentified, genetic defect in addition to the p53 mutation.

Polybrene/DMSO-assisted gene transfer. Generating stable transfectants with nanogram amounts of DNA.

Polybrene/DMSO-assisted gene transfer is a simple and versatile transfection strategy capable of producing high numbers of stable transfectants from adherent monolayer cultures with low (nanogram) quantities of exogenous DNA. The procedure involves two stages: adsorption and internalization. The former is mediated by polybrene (a polycation polymer) and favors the uniform coating of target cells with polybrene-DNA complexes. Following adsorption, the cells are permeabilized by a brief exposure to dimethyl sulfoxide (DMSO) to facilitate the uptake of DNA complexes. Diverse cell types can be exposed to a wide range of polybrene concentrations without adverse effects. By contrast, the key determinant of success is the DMSO permeabilization regime, which must be configured independently for each cell line. Protocols optimized for gene transfer in murine and human fibroblasts are presented along with a guide for the rapid optimization of the method. The advantages and limitations of the method are also discussed.

Bidirectional modulation of TNF-alpha production by alveolar macrophages in asbestos-induced pulmonary fibrosis.

Bronchoalveolar lavage (BAL) cells were isolated from rats 1, 3, and 6 weeks after a single intratracheal instillation of saline, UICC chrysotile asbestos (5 mg), or silica (5 mg). In asbestos-exposed rats, the pulmonary response was characterized by a significant increase in the number of alveolar macrophages (AMs) and the appearance of fibrotic lesions within 1 week. By contrast, mixed macrophage and neutrophil accumulations were observed in the silica group without evidence of fibrosis. Tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS)-stimulated BAL cells from asbestos-treated rats was significantly lower than controls 1 and 3 weeks after exposure. However, by 6 weeks higher levels of TNF-alpha production were noticeable in this group. Decreases in LPS-induced TNF-alpha production were also observed with BAL cells from silica-treated animals at all time points studied. Lower levels of TNF-alpha were not related to decreased BAL cell viability or the presence of a significant proportion of neutrophils in the silica group. Furthermore, biphasic changes in TNF-alpha production seen in the asbestos group were correlated with concomitant decreases (3 weeks) and increases (6 weeks) in levels of TNF-alpha mRNA in AMs. These data indicate that lower levels of TNF-alpha resulted from inhibition at the gene expression level and provide evidence for bidirectional modulation of TNF-alpha production by AMs during inflammatory reactions.

Differential expression and stability of foreign genes introduced into human fibroblasts by nuclear versus cytoplasmic microinjection.

We have compared cytoplasmic- and nuclear-delivered, glass needle-mediated microinjection protocols for their ability to support both transient and stable phenotypic expression of reporter gene constructs in non-immortalized human skin fibroblasts cultures. Microinjection of form I (covalently closed circular, supercoiled) plasmid pMC38 DNA into the nucleus of human cells resulted in high levels of transiently expressed p110gag-myc oncoprotein as detected by immunofluorescence microscopy. Likewise, the nuclear delivery of a plasmid construct bearing the entire simian virus 40 genome induced the formation of morphologically transformed foci in approximately 6% of the recipient cell population. In contrast, the introduction of plasmid DNA by the cytoplasmic route proved virtually incapable of supporting either transient gene expression or morphological transformation. In situ autoradiography of cells injected with 3H-labelled plasmid DNA revealed that whereas the material delivered directly into the nucleus was retained by this subcellular compartment for prolonged times (greater than or equal to 48 h), the radiolabelled DNA molecules introduced via the cytoplasmic route did not reach the nucleus and appeared to be substantially degraded within 8 h following injection. These results indicate unequivocally that nuclear injection is the route of choice when monitoring foreign gene expression in human cells.