The gene encoding cytosine deaminase (CD) has been expressed in the human colorectal carcinoma cell line WiDr. Metabolism studies confirm that tumor cells expressing CD convert the very nontoxic prodrug 5-fluorocytosine (5FCyt) to 5-fluorouracil (5FUra) and 5FUra metabolites. Tumor xenografts composed of CD-expressing cells can selectively generate tumor levels of >400 iAM 5FUra when the host mouse is dosed with nontoxic levels of 5FCyt. The selective metabolic conversion of 5FCyt to 5FUra in CD-expressing tumor cells results in the inhibition of thymidylate synthase and incorporation of 5FUra into RNA. 5FUra is also liberated into the surrounding environment when CD-expressing tumor cells are treated with 5FCyt. The liberated 5FUra is able to kill neighboring, non-CD-expressing tumor cells in vitro and in vivo.Most importantly, when only 2% of the tumor mass contains CD-expressing cells (98% non-CD-expressing cells), significant regresions in all tumors are observed when the host mouse is dosed with nontoxic levels of 5FCyt.We have previously reported our efforts to develop a therapeutic approach using gene transfer technology for the treatment of primary and metastatic tumors (1-4). This approach, virus-directed enzyme/prodrug therapy, exploits transcriptional differences between normal and neoplastic cells to selectively kill cancer cells. An artificial chimeric gene is created that is composed of tissue-specific transcriptional regulatory sequences linked to the coding domain of a nonmammalian enzyme. The nonmammalian enzyme can metabolically activate a nontoxic prodrug to a cytotoxic anabolite. Ifthe tissue-specific regulatory sequences are from a tumor-associated marker gene such as the carcinoembryonic antigen gene (5, 6), the prostate-specific antigen gene (7,8), or the a-fetoprotein gene (9, 10), then the artificial chimeric gene will result in tumor-specific expression of the nonmammalian enzyme and, consequently, in tumor-specific production of the cytotoxic metabolite.A critical issue influencing the therapeutic benefit of this approach will be the efficiency of gene transfer into a solid tumor. However, the efficacy of this gene therapy approach is dependent not only on the efficiency of gene transfer but also on the enzyme/prodrug system. Various enzyme/prodrug systems have been reported (1)(2)(3)(11)(12)(13)(14). A prodrug that is metabolically converted into a toxic anabolite that is not readily diffusible from one tumor cell into another will require very high gene transfer efficiency. However, if the toxic anabolite is readily diffusible, then the efficiency of gene transfer into the tumor mass may be quite low but still be able to achieve a significant therapeutic effect. Cells were lysed by freeze-thawing and centrifuged for 10 min at 8000 x g. The conditioned medium and the cell extract supernatant were evaporated to dryness, dissolved in deionized water (1 ml for medium; 100 !A for extract). Aliquots (20 1.l) of each sample were analyzed by HPLC on a Partisphere C18 column (4.6 mm x...

TnWO insertions were selected on the basis of resistance to the lipopolysaccharide (LPS)-specific bacteriophage U3. The majority of these were located in a 2-kilobase region within the rfa locus, a gene cluster of about 18 kb that contains genes for LPS core biosynthesis. The rfa::TnlO insertions all exhibited a deep rough phenotype that included hypersensitivity to hydrophobic antibiotics, a reduction in major outer membrane proteins, and production of truncated LPS. These mutations were complemented by a Clarke-Carbon plasmid known to complement rfa mutations of Salmonella typhimurium, and analysis of the insert from this plasmid showed that it contained genes for at least six polypeptides which appear to be arranged in the form of a complex operon. Defects in two of these genes were specifically implicated as the cause of the deep rough phenotype. One of these appeared to be rfiaG, which encodes a function required for attachment of the first glucose residue to the heptose region of the core. The other gene did not appear to be directly involved in determination of the sugar composition of the core. We speculate that the product of this gene is involved in the attachment of phosphate or phosphorylethanolamine to the core and that it is the lack of one of these substituents which results in the deep rough phenotype.

The 5' sequences from the human carcinoembryonic antigen gene (CEA) were analyzed using luciferase reporter gene assays. This analysis identified important cis-acting sequences needed for selective expression in CEA-positive cells. Over 50 CEA/luciferase reporter clones were constructed and analyzed in two CEA-positive and two CEA-negative cell lines. The CEA sequences analyzed extended from the translational start to 14.5 kb 5' of the CEA gene. A 408-bp region from the CEA 3' untranslated region was also examined for its effect on reporter gene activity. The CEA promoter was located between bases -90 and +69 of the transcriptional start site. Sequences between -41 and -18 were essential for expression from the CEA promoter. Multimerization of sequences between -89 and -40 resulted in copy number-related increases in both expression level and selectivity for CEA-positive cells. Two upstream regions of CEA, -13.6 to -10.7 kb or -6.1 to -4.0 kb, when linked to the multimerized promoter led to high-level, selective expression in CEA-positive cell lines. Several CEA/luciferase constructs demonstrated 80- to 120-fold higher expression in CEA-positive cell lines compared to expression in CEA-negative Hep3B cells. The expression from these constructs was quite strong in CEA-positive cells, being two- to four-fold higher than an SV40 enhancer/promoter construct. The most promising CEA transcriptional regulatory sequences were used to regulate the expression of cytosine deaminase (CD) in stable cell lines. The expression of CD was assessed directly by an enzymatic assay and indirectly by determining the in vitro IC50 to 5-fluorocytosine (5FC). The chimeric gene pCEA/CD-145 displayed the desired expression spectrum--high-level expression in the CEA-positive cells and low-level expression in CEA-negative cells. CD expression from this chimera correlated well with the expression of the endogenous CEA gene. Treatment of mice bearing NCI H508 pCEA/CD-145 tumor xenografts with 5FC lead to significant antitumor effects in vivo. The CEA/CD chimeric gene should be useful for tumor-specific suicide gene therapy of CEA-positive tumors.

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Identification of inexpensive and technically simple immunological tests useful in predicting the progression to AIDS in human immunodeficiency virus (HIV)-infected patients would be especially welcome in developing countries, in which 80% of the HIV-infected patients reside and health budgets are low. In the current study, we evaluated CD4 ؉ and total lymphocyte counts and the concentrations in serum of ␤ 2-microglobulin, p24 antigen, and immunoglobulin A (IgA) as predictors of disease progression in 74 Panamanian HIV-positive patients and 50 HIV-negative healthy individuals. Total lymphocyte and CD4 ؉-cell counts for AIDS patients (1,451 ؎ 811 cells/l, P < 0.001, and 238 ؎ 392 cells/l, P < 0.0001, respectively) and asymptomatic patients (2,393 ؎ 664 cells/l, P > 0.05, and 784 ؎ 475 cells/l, P < 0.001, respectively) were lower than those observed for healthy subjects (2,596 ؎ 631 cells/l and 1,120 ؎ 296 cells/l, respectively). The levels of ␤ 2-microglobulin and IgA in serum were significantly elevated in patients with AIDS (5.7 ؎ 3.6 mg/liter, P < 0.0001, and 541 ؎ 265 mg/dl, P < 0.0001, respectively) and asymptomatic infected subjects (3.4 ؎ 2.1 mg/liter, P ‫؍‬ 0.001, and 436 ؎ 216 mg/dl, P < 0.0001, respectively) compared with the levels in healthy subjects (2.2 ؎ 0.7 mg/liter and 204 ؎ 113 mg/dl, respectively). Nonstatistically significant differences (P > 0.05) for concentrations of p24 antigen between asymptomatic infected patients (29 ؎ 13 pg/ml) and AIDS patients (40 ؎ 23 pg/ml) were observed. Total lymphocyte counts of 1,750 cells/l or less, CD4 counts of 200 cells/l or less, ␤ 2-microglobulin concentrations in serum of 4 mg/liter or higher, concentrations of IgA in serum of 450 mg/dl or higher, and the presence in serum of p24 antigen were correlated with elevated risks for developing AIDS. Monitoring both total lymphocytes and ␤ 2-microglobulin identified 91% of the AIDS patients; these assays may allow reductions in the annual number of CD4 ؉-cell evaluations and the costs associated with monitoring the immune status of HIV-positive patients.