Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about approximately 30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7--14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5'hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.

H-cadherin expression inhibits in vitro invasiveness and tumor formation in vivo.

H-cadherin is a newly characterized cadherin molecule whose expression is decreased in a variety of human carcinoma cells, suggesting that it may play a role in maintaining normal cellular phenotype. To investigate how re-expression of H-cadherin could influence the malignant phenotype of human breast carcinoma cells in vivo, we transfected both control and H-cadherin expression vectors into human breast cancer cells (MDAMB435), which do not express H-cadherin constitutively. We found that invasiveness of these cells could be prevented by transfection with H-cadherin. We also compared the ability of control- and H-cadherin-transfected cells to induce subcutaneous tumors after injection into mammary fat pads of nude mice. Our results show that H-cadherin transfection produced a marked inhibition of tumor growth and modified the morphology of tumor cells: tumors from mice injected with control cells were significantly larger and contained larger cells having a higher degree of pleomorphism than those of tumors generated from carcinoma cells expressing H-cadherin. Altogether, these results indicate that H-cadherin expression antagonizes tumor growth in nude mice, presumably by enhancing cell-cell association in a tissue environment. These findings strongly suggest that H-cadherin could provide a possible target for corrective gene therapy against breast cancer.

Identification and partial characterization of a novel membrane-associated protein (MAP17) up-regulated in human carcinomas and modulating cell replication and tumor growth.

Using the differential display technique, we have recently reported the identification of a novel gene originally designated DD96. As determined by Northern blot and in situ hybridization, DD96 was expressed at significant levels only in a single epithelial cell population, the proximal tubular epithelial cells of the kidney. However, it was diffusely expressed in various carcinomas originating from kidney, colon, lung, and breast. Using a specific polyclonal antibody, we have not determined that the DD96 protein product is a 17-kd membrane-associated protein, which we have therefore redesignated MAP17. In normal tissues, MAP17 is expressed in significant amounts only in the kidney, where it was localized to the brush border of proximal tubular epithelial cells. However, MAP17 is expressed abundantly in carcinomas arising from kidney, colon, lung, and breast, in some cases with a membrane-associated apical glandular distribution. In tissue culture, MAP17 was localized to the cell membrane in areas of cell-cell contact, ie, the distribution of cell-function-associated proteins. Transfection of a full-length wild-type DD96 cDNA clone into a colon carcinoma cell line, HT-29, markedly decreased cell proliferation in vitro and tumor growth in vivo. Although the precise function of MAP17 remains to be determined, our findings suggest that this protein may play an important role in tumor biology.

Identification of a novel gene, selectively up-regulated in human carcinomas, using the differential display technique.

Using the differential display technique, selecting for genes up-regulated in renal cell carcinoma compared with normal renal parenchyma, we isolated a novel gene, designated DD96. As determined by in situ and Northern blot hybridization studies, DD96 is expressed only in rare normal epithelial cell populations, such as the proximal tubular epithelial cells of the kidney. However, it is expressed diffusely in malignant epithelial cells of the wide majority of renal cell carcinomas. In addition, DD96 is overexpressed markedly in various human carcinomas originating from the colon, breast, and lung, as well as in a number of cell lines derived from tumors of these organs compared with normal epithelial cell populations. Furthermore, the expression of DD96 is induced in immortalized breast ductal epithelial cell lines compared with normal breast ductal epithelial cells, and, in vivo, in premalignant conditions, such as adenoma of the colon and ductal carcinoma in situ of the breast. Sequence analysis of a complete cDNA clone isolated from a human kidney cDNA library revealed that DD96 encodes for a protein of approximately Mr 13,500. These results suggest that DD96 may play a role in the early events associated with malignant transformation; however, its function remains to be determined.