D. Margaret Hunt

Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/neu positive tumor cell lines.

Silencing of a specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. We designed 3 such siRNA against the Her2/neu (HER2) gene. The HER2 gene is known to play an important role in the oncogenesis of several types of cancers, such as breast, ovarian, colon and gastric cancers. Introduction of the siRNA into HER2 positive tumor lines in vitro greatly reduced the cell surface expression of the HER2 protein. Concurrently, a range of effects on cell physiology, such as growth inhibition or apoptosis, was observed. The expression of HLA class I was observed to be upregulated when HER2 was silenced with siRNA. Treatment of SKBr3 and MCF7/HER2 tumor cell lines with the HER2 siRNA resulted in growth arrest of cells in the late G1/S‐phase. Our results suggest that siRNA may be an effective method of abrogating the effect of HER2 in tumorigenesis.

A Single Amino Acid Change in the L-Polymerase Protein of Vesicular Stomatitis Virus Completely Abolishes Viral mRNA Cap Methylation

ABSTRACT The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5′-cap structures methylated at the guanine-N7 and 2′-O-adenosine positions (7mGpppAm). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the L-polymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5′-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-l-methionine-binding domain of the L protein.

Hemopexin in the human retina: protection of the retina against heme-mediated toxicity.

The existence of the blood‐retinal barrier means that proteins that protect the retina from damage by reactive oxygen species must either be made locally or specifically transported across the barrier cells; however, such transepithelial transport does not seem to occur. Among the circulatory proteins that protect against iron‐catalyzed production of free radicals are apo‐transferrin, which binds ferric iron and has previously been shown to be made by cells of the neural retina (Davis and Hunt, 1993, J. Cell Physiol., 156:280–285), and the extracellular antioxidant, apo‐hemopexin, which binds free heme (iron‐protoporphyrin IX). Since hemorrhage and heme release can be important contributing factors in retinal disease, evidence of a hemopexin‐based retinal protection system was sought. The human retina has been shown to contain apo‐hemopexin which is probably synthesized locally since its mRNA can be detected in retinal tissue dissected from human donor eyes. It is likely that the retina contains a mechanism for the degradation of hemopexin‐bound heme since the blood‐retinal barrier also precludes the exit of heme‐hemopexin from the retina. Retinal pigment epithelial cells have been found to bind and internalize heme‐hemopexin in a temperature‐dependent, saturable, and specific manner, analogous to the receptor‐mediated endocytic system of hepatoma cells. Moreover, the binding of heme‐hemopexin to the cells stimulates the expression of heme oxygenase‐1, metallothionein‐1, and ferritin.

Amplification of stromelysin-3 transcripts from carcinomas of the colon☆

Stromelysin-3 has been recently described in association with the stroma of different types of cancer including colorectal carcinomas. This article reports the detection of transcripts for stromelysin-3 (matrix metalloproteinase-11 [MMP-11]) in extracts of tissue from colorectal carcinomas using the technique of reverse transcription-polymerase chain reaction (RT-PCR). In 12 cases of primary colon carcinoma, stromelysin-3 messenger RNA (mRNA) was detected after 25 cycles, whereas this procedure did not reveal stromelysin-3 mRNA expression in one rectal carcinoma micrometastasis to the liver or in normal colon tissue (controls) after 30 cycles of PCR. However, stromelysin-3 mRNA was detected in normal colon specimens after 45 cycles. The high sensitivity of this technique allows application for the investigation of the expression of stromelysin-3 in small amounts of tissue.

Increased synthesis of polycistronic mRNA associated with increased polyadenylation by vesicular stornatitis virus

Electron microscopy suggested that the mRNA produced in vitro by tsG16(I), a temperature-sensitive mutant of vesicular stomatitis virus, contained an increased proportion of polycistronic mRNAs. Using hybrid selection, we found that the poly(A)+ mRNA synthesized in vitro by tsG16(I) contained approximately two to three times more polycistronic mRNA than did poly(A)+ mRNA synthesized in vitro by the parental wild-type (wt) virus. The increase in polycistronic mRNA occurred at all intergenic junctions examined. In vitro, tsG16(I) has an increased polyadenylation phenotype and a temperature-sensitive transcriptase activity that appear to be due to different mutations. Partial revertants of tsG16(I), which have lost the aberrant polyadenylation phenotype but retain the in vitro thermosensitive transcriptase, produced wt amounts of polycistronic mRNA. This suggested that the increased production of polycistronic mRNA by tsG16(I) may be associated with the increased polyadenylation phenotype of this mutant. These data further support the hypothesis that an increase in size of poly(A) tracts is associated with increased production of polycistronic mRNA.

Revertants of a mutant of vesicular stomatitis virus which has an aberrant polyadenylation activity and a temperature-sensitive transcriptase

tsG16(l), a temperature-sensitive mutant of vesicular stomatitis virus, in vitro has at least three phenotypic differences from its parental wild-type (wt) virus due to mutation of the L gene. It was not known whether (i) the temperature-sensitivity of the transcriptase, (ii) the aberrant polyadenylation phenotype, and (iii) the extent of increased polyadenylation in response to S-adenosylhomocysteine (SAH) were associated with a single mutation. Spontaneous partial revertants were selected from tsG16(I) on the basis of the ability to form plaques at 34.7 degrees (35G16 revertants) or from 35G16 revertants on the basis of the ability to form plaques at 37 degrees (37G16 revertants). All six 35G16 revertants had fully (five) or partially (one) recovered the wt polyadenylation phenotype and the former five had also fully recovered the wt polyadenylation response to SAH. This suggested that a single mutation in tsG16(I) was probably associated with both of these phenotypes and also probably conferred the inability to grow at 34.7 degrees. None of the 35G16 revertants regained the wt phenotype for thermosensitivity of the transcriptase, although both of the 37G16 revertants did. This suggested that in vitro temperature-sensitivity of transcription by tsG16(I) might be due to a mutation different than the one affecting polyadenylation in the absence or presence of SAH.