Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks, alkylations, or x-ray damage.

The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plus-Giemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions.

Cloning of a novel receptor expressed in rat prostate and ovary.

We have cloned a novel member of the nuclear receptor superfamily. The cDNA of clone 29 was isolated from a rat prostate cDNA library and it encodes a protein of 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Clone 29 protein is unique in that it is highly homologous to the rat estrogen receptor (ER) protein, particularly in the DNA-binding domain (95%) and in the C-terminal ligand-binding domain (55%). Expression of clone 29 in rat tissues was investigated by in situ hybridization and prominent expression was found in prostate and ovary. In the prostate clone 29 is expressed in the epithelial cells of the secretory alveoli, whereas in the ovary the granuloma cells in primary, secondary, and mature follicles showed expression of clone 29. Saturation ligand-binding analysis of in vitro synthesized clone 29 protein revealed a single binding component for 17beta-estradiol (E2) with high affinity (Kd= 0.6 nM). In ligand-competition experiments the binding affinity decreased in the order E2 > diethylstilbestrol > estriol > estrone > 5alpha-androstane-3beta,17beta-diol >> testosterone = progesterone = corticosterone = 5alpha-androstane-3alpha,17beta-diol. In cotransfection experiments of Chinese hamster ovary cells with a clone 29 expression vector and an estrogen-regulated reporter gene, maximal stimulation (about 3-fold) of reporter gene activity was found during incubation with 10 nM of E2. Neither progesterone, testosterone, dexamethasone, thyroid hormone, all-trans-retinoic acid, nor 5alpha-androstane-3alpha,I7beta-diol could stimulate reporter gene activity, whereas estrone and 5alpha-androstane-3beta,17beta-diol did. We conclude that clone 29 cDNA encodes a novel rat ER, which we suggest be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.

Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants.

A group of resident ER proteins have been identified that are proposed to function as molecular chaperones. The best characterized of these is BiP/GRP78, an hsp70 homologue that binds peptides containing hydrophobic residues in vitro and unfolded or unassembled proteins in vivo. However, evidence that mammalian BiP plays a direct role in protein folding remains circumstantial. In this study, we examine how BiP interacts with a particular substrate, immunoglobulin light chain (lambda LC), during its folding. Wild-type hamster BiP and several well-characterized BiP ATPase mutants were used in transient expression experiments. We demonstrate that wild-type lambda LCs showed prolonged association with mutant BiP which inhibited their secretion. Both wild-type and mutant BiP bound only to unfolded and partially folded LCs. The wild-type BiP was released from the incompletely folded LCs, allowing them to fold and be secreted, whereas the mutant BiP was not released. As a result, the LCs that were bound to BiP mutants were unable to undergo complete disulfide bond formation and were retained in the ER. Our experiments suggest that LCs undergo both BiP-dependent and BiP-independent folding steps, demonstrating that both ATP binding and hydrolysis activities of BiP are essential for the completion of LC folding in vivo and reveal that BiP must release before disulfide bond formation can occur in that domain.

Purification and cDNA cloning of a second apoptosis-related cysteine protease that cleaves and activates sterol regulatory element binding proteins.

We have purified from hamster liver a second cysteine protease that cleaves and activates sterol regulatory element binding proteins (SREBPs). cDNA cloning revealed that this enzyme is the hamster equivalent of Mch3, a human enzyme that is related to the interleukin 1beta converting enzyme. We call this enzyme Mch3/SCA-2. It is 54% identical to hamster CPP32/SCA-1, a cysteine protease that was earlier shown to cleave SREBPs at a conserved Asp between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. This cleavage liberates an NH2-terminal fragment of approximately 460 amino acids that activates transcription of genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. Mch3/SCA-2 and CPP32/SCA-I are synthesized as inactive 30-35 kDa precursors that are thought to be cleaved during apoptosis to generate active fragments of approximately 20 and approximately 10 kDa. The current data lend further support to the notion that SREBPs are cleaved and activated as part of the program in programmed cell death.

Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform.

Prion diseases are a group of fatal neurodegenerative disorders that are unique in being infectious, genetic, and sporadic in origin. Infectious cases are caused by prions, which are composed primarily of PrPSc, a posttranslationally modified isoform of the normal cellular prion protein PrPC. Inherited cases are linked to insertional or point mutations in the host gene encoding PrPC. To investigate the molecular mechanisms underlying inherited prion diseases, we have constructed stably transfected Chinese hamster ovary cells that express mouse PrPs homologous to two human PrPs associated with familial Creutzfeldt-Jakob disease. One mouse PrP molecule carries a Glu-->Lys substitution at codon 199, and the other carries an insertion of six additional octapeptide repeats between codons 51 and 90. We find that both of these mutant PrPs display several biochemical hallmarks of PrPSc when synthesized in cell culture. Unlike wild-type PrP, the mutant proteins are detergent insoluble and are relatively resistant to digestion by proteinase K, yielding an N-terminally truncated core fragment of 27-30 kDa. Pulse-chase labeling experiments demonstrate that these properties are acquired posttranslationally, and are accompanied by increased metabolic stability of the protein. Our results provide the first evidence that a molecule with properties reminiscent of PrPSc can be generated de novo in cultured cells.

Potentiation of the bioavailability of daidzin by an extract of Radix puerariae.

The dose effect of pure daidzin on the suppression of ethanol intake in Syrian golden hamsters was compared with that of crude daidzin contained in a methanol extract of Radix puerariae (RP). EC50 values estimated from the graded dose-response curves for pure daidzin and RP extract daidzin are 23 and 2.3 mg per hamster per day, respectively. Apparently the antidipsotropic activity of the RP extract cannot be accounted for solely by its daidzin content (22 mg/g). In addition to daidzin, six other isoflavones were identified in the RP extract and quantified--namely, puerarin (160 mg per g of extract), genistin (3.7 mg/g), daidzein (2.6 mg/g), daidzein-4',7-diglucoside (1.2 mg/g), genistein (0.2 mg/g), and formononetin (0.16 mg/g). None of these, administered either alone or combined, contributes in any significant way to the antidipsotropic activity of the extract. Plasma daidzin concentration-time curves determined in hamsters administered various doses of pure daidzin or RP extract by i.p.injection indicate that the crude extract daidzin has approximately 10 times greater bioavailability than the pure compound. Reconstruction of the dose-response effects for pure and crude daidzin using bioavailable daidzin rather than administered dose gives a single curve. Synthetic daidzin added to the RP extract acquires the bioavailability of the endogenous daidzin that exists naturally in the extract. These results show that (i) daidzin is the major active principle in methanol extracts of RP, and (ii) additional constituents in the methanol extract of RP assist uptake of daidzin in golden hamsters.

Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene.

The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication-dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors.

Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy.

Treatment of a human breast cancer cell line (MDA-MB-435) in nude mice with a recombinant adenovirus containing the human interferon (IFN) consensus gene, IFN-con1 (ad5/IFN), resulted in tumor regression in 100% of the animals. Tumor regression occurred when virus was injected either within 24 hr of tumor cell implantation or with established tumors. However, regression of the tumor was also observed in controls in which either the wild-type virus or a recombinant virus containing the luciferase gene was used, although tumor growth was not completely suppressed. Tumor regression was accompanied by a decrease in p53 expression. Two other tumors, the human myelogenous leukemic cell line K562 and the hamster melanoma tumor RPMI 1846, also responded to treatment but only with ad5/IFN. In the case of K562 tumors, there was complete regression of the tumor, and tumors derived from RPMI 1846 showed partial regression. We propose that the complete regression of the breast cancer with the recombinant virus ad5/IFN was the result of two events: viral oncolysis in which tumor cells are being selectively lysed by the replication-competent virus and the enhanced effect of expression of the IFN-con1 gene. K562 and RPMI 1846 tumors regressed only as a result of IFN gene therapy. This was confirmed by in vitro analysis. Our results indicate that a combination of viral oncolysis with a virus of low pathogenicity, itself resistant to the effects of IFN and IFN gene therapy, might be a fruitful approach to the treatment of a variety of different tumors, in particular breast cancers.

Protein-free cell culture on an artificial substrate with covalently immobilized insulin.

Insulin was immobilized on a surface-hydrolyzed poly(methyl methacrylate) film. Chinese hamster ovary cells overexpressing human insulin receptors were cultured on the film in the absence of serum or soluble proteins. Small amounts of immobilized insulin (1-10% of the required amount of free insulin) were sufficient to stimulate cell proliferation. In addition, the maximal mitogenic effect of immobilized insulin was greater than that of free insulin. Immobilized insulin activated the insulin receptor and downstream signaling proteins, and this activation persisted for longer periods than that obtained with free insulin, probably explaining the greater mitogenic effect of the immobilized insulin. Finally the immobilized-insulin film was usable repeatedly without marked loss of activity.

Regulation of meiotic chromatin loop size by chromosomal position.

At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging.

Activation of CD4+ T lymphocytes form interleukin 2-deficient mice by costimulatory B7 molecules.

Interleukin 2 (IL-2)-deficient (IL-2-/-) mice develop hemolytic anemia and chronic inflammatory bowel disease. Importantly, the induction of disease in IL-2-deficient mice is critically dependent on CD4+ T cells. We have studied the requirements of T cells from IL-2-deficient mice for costimulation with B7 antigens. Stable B7-1 or B7-2 chinese hamster ovary (CHO) cell transfectants could synergize with anti-CD3 monoclonal antibody (mAb) to induce the proliferation of CD4+ T cells from IL-2-/- mutant mice. Further mechanistic studies established that B7-induced activation resulted in surface expression of the alpha chain of the IL-2 receptor. B7-induced proliferation occurred independently of IL-4 and was largely independent of the common gamma chain of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors. Finally, anti-B7-2 but not anti-B7-1 mAb was able to inhibit the activation of IL-2-/- T cells induced by anti-CD3 mAb in the presence of syngeneic antigen-presenting cells. The results of our experiments indicate that IL-2-/- CD4+ T cells remain responsive to B7 stimulation and raise the possibility that B7 antagonists have a role in the prevention/treatment of inflammatory bowel disease.

Bombesin antagonist prevents CO2 laser-induced promotion of oral cancer.

We previously reported that CO2 laser incisions in carcinogen-initiated fields promoted cancer development and caused release of growth factors. Here we examined the quantitative and additive properties of this tumor-promoting event and examined whether this promotion could be nullified by treatment with a bombesin antagonist, which down-regulates epidermal growth factor receptors. The model used for cancer promotion was the hamster buccal cheek pouch that had been treated with a carcinogen (9,10-dimethyl-1,2-benzanthracene) for 6 weeks, producing premalignant lesions. These lesions would evolve into a cancer eventually without further treatment. Promotion was measured both by increased fluorescence in response to systemically administered Photofrin, measured noninvasively using an in vivo fluorescence photometer, and by the timing of appearance of clinical tumors. Laser incisions (0-3) were made into the hamster cheek 1 week apart, or three incisions were done 1 day apart. Another group of animals received bombesin antagonist RC-3095 for 4 weeks during the time incisions were made, again measuring promotion. Laser incisions 1 week apart produced additive promotion, whereas three incisions 1 day apart were not statistically different from the group receiving only one incision. RC-3095 treatment completely eliminated the promoting effects of incision and totally stopped promotion for the 4-week period of treatment. After discontinuing treatment with RC-3095, lesion progression resumed at the untreated control rate. This work confirms that the promoting event of a laser incision follows a comparable time course to release of growth factors after such an incision and that it can be eliminated by treatment with bombesin antagonists.

Mutagenic specificity of solar UV light in nucleotide excision repair-deficient rodent cells.

To investigate the role of nucleotide excision repair (NER) in the cellular processing of carcinogenic DNA photoproducts induced by defined, environmentally relevant portions of the solar wavelength spectrum, we have determined the mutagenic specificity of simulated sunlight (310-1100 nm), UVA (350-400 nm), and UVB (290-320 nm), as well as of the "nonsolar" model mutagen 254-nm UVC, at the adenine phosphoribosyltransferase (aprt) locus in NER-deficient (ERCC1) Chinese hamster ovary (CHO) cells. The frequency distributions of mutational classes induced by UVB and by simulated sunlight in repair-deficient CHO cells were virtually identical, each showing a marked increase in tandem CC-->TT transitions relative to NER-proficient cells. A striking increase in CC-->TT events was also previously documented for mutated p53 tumor-suppressor genes from nonmelanoma tumors of NER-deficient, skin cancer-prone xeroderma pigmentosum patients, compared to normal individuals. The data therefore indicate that the aprt gene in NER-deficient cultured rodent cells irradiated with artificial solar light generates the same distinctive "fingerprint" for sunlight mutagenesis as the p53 locus in NER-deficient humans exposed to natural sunlight in vivo. Moreover, in strong contrast to the situation for repair-component CHO cells, where a significant role for UVA was previously noted, the mutagenic specificity of simulated sunlight in NER-deficient CHO cells and of natural sunlight in humans afflicted with xeroderma pigmentosum can be entirely accounted for by the UVB portion of the solar wavelength spectrum.

Proposed active site domain in estrogen sulfotransferase as determined by mutational analysis.

Point mutations were selectively introduced into a cDNA for guinea pig estrogen sulfotransferase (gpEST); each construct was then expressed in Chinese hamster ovary K1 cells. The molecular site chosen for study is a conserved GXXGXXK sequence that resembles the P-loop-type nucleotide-binding motif for ATP- and GTP-binding proteins and is located near the C terminus of all steroid and phenol(aryl) sulfotransferases for which the primary structures are known. Preliminary experiments demonstrated that the GXXGXXK motif is essential for binding the activated sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The present study was undertaken to ascertain the relative importance of each individual residue of the motif. While the mutation of a single motif residue had little effect on the interaction between gpEST and PAPS as determined by kinetic analysis and photoaffinity labeling, the mutation of any two residues in concert resulted in an approximate 10-fold increase in the Km for PAPS and reduced photoaffinity labeling. The mutation of all three motif residues resulted in an inactive enzyme and complete loss of photoaffinity labeling. Interestingly, several mutants also displayed a striking effect on the Km for the steroid substrate; double mutants, again, demonstrated greater perturbations (8- to 28-fold increase) than did single mutants. Unexpectedly, whereas the mutation of nonmotif residues had a negligible effect on the Km for PAPS, a marked increase in the Km for the estrogen substrate ( > 30-fold) was noted. On the basis of these findings, it is concluded that the sequence GISGDWKN within the C-terminal domain of gpEST represents a critical component of the active site.

Regulation of microfilament organization and anchorage-independent growth by tropomyosin 1.

Variants of chemically immortalized Syrian hamster embryo cells that had either retained (supB+) or lost (supB-) the ability to suppress tumorigenicity when hybridized with a fibrosarcoma cell line were subcloned. Both supB cell types are nontumorigenic; however, the supB- but not supB+ cells exhibit conditional anchorage-independent growth. Alterations of actin microfilament organization were observed in supB- but not supB+ cells that corresponded to a significant reduction of the actin-binding protein tropomyosin 1 (TM-1) in subB- cells. To examine the possibility of a direct relationship between TM-1 expression and the subB- phenotype, subB+ cells were transfected with an expression vector containing the TM-1 cDNA in an antisense orientation. The antisense-induced reduction of TM-1 levels in supB+ clones caused a microfilament reorganization and conferred anchorage-independent growth potential that were indistinguishable from those characteristic of supB- cells. These data provide direct evidence that TM-1 regulates both microfilament organization and anchorage-independent growth and suggest that microfilament alterations are sufficient for anchorage-independent growth.