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p62 is a RNA-binding protein that was isolated by immunoscreening a cDNA expression library with autoantibodies from patients with hepatocellular carcinoma (HCC). This autoantigen binds to mRNA encoding insulin-like growth factor II, which has been found to be overexpressed in HCC and is tumorigenic in transgenic animals. Immunohistochemical analysis of HCC liver showed that 33% (9 of 27) exhibited readily detectable staining of p62 protein in the cytoplasm of all malignant cells in cancer nodules, whereas it was undetectable in adjacent nonmalignant liver cells. In addition one of two patients with cholangiocarcinoma expressed p62 in malignant bile duct epithelial cells. p62 expression was also detected in scattered cells in cirrhotic nodules in contrast to uniform expression in all cells in HCC nodules. In HCC nodules, p62 mRNA was also detected by reverse transcriptase-polymerase chain reaction analysis. Nine normal adult livers did not contain detectable p62 mRNA or p62 protein whereas five fetal livers were all positive for mRNA and protein. The observations show that p62 is developmentally regulated, expressed in fetal, but not in adult liver, and aberrantly expressed in HCC and could be playing a role in abnormal cell proliferation in HCC and cirrhosis by modulating expression of growth factors such as insulin-like growth factor II.

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (Bm-NPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.

Protein IE1 is the product of a baculovirus gene, ie1, that is activated immediately upon entrance of the viral genome into the cell nucleus. This protein was previously shown to be a trans-regulator of viral genes whose products are required for initiation of the infectious cycle including viral DNA replication. To test whether the IE1 protein is also capable of trans-regulating nuclear genes of the host in vitro and in vivo, we transfected the ie1 gene of Bombyx mori nuclear polyhedrosis virus (BmNPV) into silkworm Bm5 tissue culture cells together with expression cassettes directing expression of chloramphenicol acetyl transferase or juvenile hormone esterase under the control of the cytoplasmic actin A3 gene promoter of B. mori. Cotransfection with the ie1 gene resulted in a dramatic increase in the amount of the two enzymes expressed in the transfected cells. The increased enzyme activities correlate with an increased accumulation of the corresponding mRNAs, and the latter is caused by an increase in the rate of transcription directed by the cytoplasmic actin gene promoter. The chromosomal cytoplasmic actin gene of Bm5 cells is also upregulated upon transfection of the cells with the ie1 gene. However, infection of cells with BmNPV does not cause an increase in the level of expression of the endogenous cytoplasmic actin gene. Thus, the effect of IE1 on the transcriptional properties of the cytoplasmic actin gene vary depending on whether IE1 is expressed in isolation or in the context of a viral infection. The trans-activating effects of BmNPV ie1 gene expression on the silkmoth actin promoter are also evident in Spodoptera frugiperda Sf21 and Choristoneura fumiferana Cf1 tissue culture cells. Finally, the ie1 gene of Autographa californica nuclear polyhedrosis virus can substitute for its BmNPV counterpart in all cell lines tested.

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