Dominant-negative polo-like kinase 1 induces mitotic catastrophe independent of cdc25C function.

Polo-like kinase 1 (PLK1), which has been shown to have a critical role in mitosis, is one possible target for cancer therapeutic intervention. PLK1, at least in Xenopus, starts the mitotic cascade by phosphorylating and activating cdc25C phosphatase. Also, loss of PLK1 function has been shown to induce mitotic catastrophe in a HeLa cervical carcinoma cell line but not in normal Hs68 fibroblasts. We wanted to understand whether the selective mitotic catastrophe in HeLa cells could be extended to other tumor types, and, if so, whether it could be attributable to a tumor-specific loss of dependence on PLK1 for cdc25C activation. When PLK1 function was blocked through adenovirus delivery of a dominant-negative gene, we observed tumor-selective apoptosis in most tumor cell lines. In some lines, dominant-negative PLK1 induced a mitotic catastrophe similar to that published in HeLa cells (K. E. Mundt et al., Biochem. Biophys Res. Commun., 239: 377-385, 1997). Normal human mammary epithelial cells, although arrested in mitosis, appeared to escape the loss of centrosome maturation and mitotic catastrophe seen in tumor lines. Mitotic phosphorylation of cdc25C and activation of cdk1 was blocked by dominant-negative PLK1 in human mammary epithelial cells as well as in the tumor lines regardless of whether they underwent mitotic catastrophe. These data strongly argue that the mitotic catastrophe is not attributable to a lack of dependence for PLK1 in activating cdc25C.

An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers.

The antisense activity and gene specificity of two classes of oligonucleotides (ONs) were directly compared in a highly controlled assay. One class of ONs has been proposed to act by targeting the degradation of specific RNAs through an RNase H-mediated mechanism and consists of C-5 propynyl pyrimidine phosphorothioate ONs (propyne-S-ON). The second class of antisense agents has been proposed to function by sterically blocking target RNA formation, transport or translation and includes sugar modified (2'-O-allyl) ONs and peptide nucleic acids (PNAs). Using a CV-1 cell based microinjection assay, we targeted antisense agents representing both classes to various cloned sequences localized within the SV40 large T antigen RNA. We determined the propyne-S-ON was the most potent and gene-specific agent of the two classes which likely reflected its ability to allow RNase H cleavage of its target. The PNA oligomer inhibited T Ag expression via an antisense mechanism, but was less effective than the propyne-S-ON; the lack of potency may have been due in part to the PNAs slow kinetics of RNA association. Interestingly, unlike the 2'-O-allyl ON, the antisense activity of the PNA was not restricted to the 5' untranslated region of the T Ag RNA. Based on these findings we conclude that PNAs could be effective antisense agents with additional chemical modification that will lead to more rapid association with their RNA target.

Strand-invasion of duplex DNA by peptide nucleic acid oligomers.

Polyamide oligomers, termed peptide nucleic acids (PNAs), bind with high affinity to both DNA and RNA and offer both antisense and antigene approaches for regulating gene expression. When a PNA binds to a complementary sequence in a double-stranded DNA, one strand of the duplex is displaced, and a stable D-loop is formed. Unlike oligodeoxynucleotides for which binding polarity is determined by the deoxyribose sugar, the unrestrained polyamide backbone of the PNA could permit binding to a DNA target in an orientation-independent manner. We now provide evidence that PNAs can, in fact, bind to their complementary sequence in DNA independent of the DNA-strand polarity--that is, a PNA binds to DNA in both "parallel" and "antiparallel" fashion. With a mixed-sequence 15-mer PNA, kinetic studies of PNA.DNA interactions revealed that D-loop formation was rapid and the complex was stable for several hours. However, when measured either by gel-mobility-shift analysis or RNA polymerase II-elongation termination, D-loop formation was salt dependent, but PNA-strand dissociation was not salt dependent. We observed that D-loop-containing DNA fragments had anomalous gel mobilities that varied as a function of the position of the D-loop relative to the DNA termini. On the basis of permutation analysis, the decreased mobility of the PNA.DNA complex was attributed to a bend in the DNA at or near the D-loop.

Antisense and antigene properties of peptide nucleic acids.

Peptide nucleic acids (PNAs) are polyamide oligomers that can strand invade duplex DNA, causing displacement of one DNA strand and formation of a D-loop. Binding of either a T10 PNA or a mixed sequence 15-mer PNA to the transcribed strand of a G-free transcription cassette caused 90 to 100 percent site-specific termination of pol II transcription elongation. When a T10 PNA was bound on the nontranscribed strand, site-specific inhibition never exceeded 50 percent. Binding of PNAs to RNA resulted in site-specific termination of both reverse transcription and in vitro translation, precisely at the position of the PNA.RNA heteroduplex. Nuclear microinjection of cells constitutively expressing SV40 large T antigen (T Ag) with either a 15-mer or 20-mer PNA targeted to the T Ag messenger RNA suppressed T Ag expression. This effect was specific in that there was no reduction in beta-galactosidase expression from a coinjected expression vector and no inhibition of T Ag expression after microinjection of a 10-mer PNA.

Characterization of a highly invasive and spontaneously metastatic human malignant melanoma cell line.

Although the incidence of, and deaths due to, malignant melanoma are rising at a rapid rate, few experimental models mimic the highly metastatic properties associated with the pathogenesis of the human disease, making study of the disease difficult. Thus, new human models are required to understand melanoma biology, especially its metastatic properties. Here we describe C8161, a highly invasive and spontaneously metastatic human melanoma cell line, which grows progressively in the subcutis of athymic nude mice with an average doubling time of approximately 6 days. By the time the tumor reaches a diameter of 1 cm, amelanotic metastases in lymph nodes, skin, peritoneal wall, spleen and lungs have formed. By comparing C8161 to variants from other well-characterized human malignant melanomas (A375 and MeWo) with differing metastatic traits, properties presumed to be involved in metastatic propensity were examined. C8161 showed a 2- to 14-fold higher ability to invade reconstituted basement membrane barriers in the MICS and correspondingly high type-IV collagenase mRNA levels and collagenolytic activity, as compared with other melanoma cell lines. Likewise, differential adhesion to immobilized RBM or HUVEC monolayers was observed, but did not correlate to rank orders of malignant properties. Recently, a correlation between surface expression of ICAM-1 and secondary tumor formation by human melanomas has been described in several laboratories. Basal levels of ICAM-1 on C8161, A375 and MeWo human melanomas were compared, but no correlation with metastatic potential was noted. Proto-oncogene expression in C8161 cells was compared with A375P and A375M variants using Northern blot analysis. c-myc expression was 6-fold greater than both A375 variants; c-fos expression was 3.4-fold less than A375P and 1.7-fold less than A375M; c-jun in C8161 cells was 2.5-fold and 2.1-fold greater than expression in A375P and A375M, respectively. Because C8161 is so highly malignant, amenable to experimental manipulation, and its behavior in nude mice mimics the clinical course of malignant melanoma, this cell line will prove valuable for studying properties associated with human melanoma tumor progression.