Scintigraphic imaging of oncogenes with antisense probes: does it make sense?

Based on the specificity of the Watson-Crick base pairing formation, antisense deoxyoligonucleotides have been used to inhibit the expression of oncogenes in various cancer cells. Activation of an oncogene by means of amplification leads to an increased, detectable amount of the mRNA transcript in the cytoplasm. The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. This work and recent developments in the antisense field lead to the expectation of a new class of radiopharmaceuticals with unique specificity.

A reappraisal of the role of insulin-like growth factor I in the regulation of human hematopoiesis.

IGF-I has been reported to increase hematopoietic progenitor cell cloning efficiency. To investigate this phenomenon, we studied the IGF-I responsiveness of human marrow cells expressing IGF-I receptor (IGF-IR), a direct strategy not used previously. IGF-IR+ and control CD34+ marrow cells were isolated using immunoaffinity methods. Then, the cells were cloned in methylcellulose containing variable amounts of serum- and lineage-appropriate growth factors supplemented with recombinant human IGF-I. In contrast to CD34+ cells, IGF-IR+ cells never gave rise to CFU-Blast, CFU-Mix, CFU-GM, BFU-E, or CFU-E. To substantiate the suggestion that CD34+ and IGF-IR+ cells were distinct populations, we used reverse transcription PCR to detect IGF-I, EpO, and KIT receptor mRNAs in these cells. The mRNA phenotype of CD34+ cells was EpO (+), KIT (+), and IGF-IR (-), while IGF-IR+ cells were IGF-IR (+), EpO (-), and KIT (-). These results suggested that IGF-IR is either not expressed or expressed at low levels on normal hematopoietic progenitor cells. Functional significance of the latter possibility was tested by exposing CD34+ cells to IGF-IR antisense oligodeoxynucleotides. Colony formation was unaffected by oligodeoxynucleotide disruption of IGF-IR, suggesting that, even if expressed at low level, the receptor's functional significance was doubtful. Nevertheless, when cultured in the presence of IGF-I, IGF-IR+ cells elaborated an activity with mild BFU-E stimulatory effects. Accordingly, if IGF-I plays a role in hematopoietic colony formation, it is probably and results from stimulation of IGF-IR-positive ancillary cells to secrete growth factors. Studies carried out with human leukemia cells yielded similar results.

Biologic and therapeutic significance of MYB expression in human melanoma.

We investigated the therapeutic potential of employing antisense oligodeoxynucleotides to target the disruption of MYB, a gene which has been postulated to play a pathogenetic role in cutaneous melanoma. We found that MYB was expressed at low levels in several human melanoma cell lines. Also, growth of representative lines in vitro was inhibited in a dose- and sequence-dependent manner by targeting the MYB gene with unmodified or phosphorothioate-modified antisense oligodeoxynucleotides. Inhibition of cell growth correlated with specific decrease of MYB mRNA. In SCID mice bearing human melanoma tumors, infusion of MYB antisense transiently suppressed MYB gene expression but effected long-term growth suppression of transplanted tumor cells. Toxicity of the oligodeoxynucleotides was minimal in mice, even when targeted to the murine Myb gene. These results suggest that the MYB gene may play an important, though undefined, role in the growth of at least some human melanomas. Inhibition of MYB expression might be of use in the treatment of this disease.

Acute- and chronic-phase chronic myelogenous leukemia colony-forming units are highly sensitive to the growth inhibitory effects of c-myb antisense oligodeoxynucleotides.

We have previously demonstrated that malignant hematopoietic colony-forming units (CFUs) may be purged from normal CFU by exposure to c-myb antisense oligodeoxynucleotides (oligomers). This novel strategy appeared particularly promising for patients with chronic myelogenous leukemia (CML) in blast crisis, since in some cases complete elimination of bcr-abl-expressing cells was accomplished. We have examined 11 additional patients, including seven in chronic phase, in order to extend these initial observations. We sought in particular to determine if elimination of bcr-abl-expressing clones was a usual event. Exposure of CML cells to c-myb antisense oligomers resulted in inhibition of CFU-granulocyte, macrophage (CFU-GM)-derived colony formation in eight of 11 (73%) cases evaluated. Inhibition was antisense sequence-specific, dose-dependent, ranged between 58% and 93%, and was statistically significant (P less than or equal to .03) in seven of the eight cases. In two cases, CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM)-derived colony formation was also examined and found to be inhibited by the c-myb antisense oligomers in a sequence-specific manner. To determine whether CML CFU had been reduced or eliminated after exposure to the antisense oligomers, we examined cells in the residual colonies for bcr-abl mRNA expression using a reverse transcription-polymerase chain reaction detection technique (RT-PCR). Eight cases were evaluated and in each case where antisense myb inhibited growth, bcr-abl expression as detected by RT-PCR was either greatly decreased or nondetectable. No residual leukemic CFU were demonstrable on replating of treated cells. These results suggest that c-myb antisense oligomers substantially inhibit the growth and survival of CML CFU in both chronic and blast phase of disease. They may therefore prove useful for both ex vivo and in vivo treatment of CML.

Role of the KIT protooncogene in normal and malignant human hematopoiesis.

The role of the KIT protooncogene in human hematopoiesis is uncertain. Therefore, we examined KIT mRNA expression in normal human bone marrow mononuclear cells (MNC) and used antisense oligodeoxynucleotides (oligomers) to disrupt KIT function. KIT mRNA was detected with certainty only in growth factor-stimulated MNC. Expression was essentially abrogated by making MNC quiescent or by inhibiting myb gene function. Oligomers blocked KIT mRNA expression in a dose-response and sequence-specific manner, thereby allowing functional examination of the KIT receptor. In experiments with either partially purified or CD34(+)-enriched MNC, neither granulocyte nor megakaryocyte colony formation was inhibited by oligomer exposure. In contrast, KIT antisense oligomers inhibited interleukin 3/erythropoietin-driven erythroid colony formation approximately 70% and "stem cell factor"/erythropoietin-driven colony formation 100%. The presence of erythroid progenitor cell subsets with differential requirements for KIT function is therefore suggested. Growth of hematopoietic colonies from chronic myeloid leukemia and polycythemia vera patients was also inhibited, while acute leukemia colony growth appeared less sensitive to KIT deprivation. These results suggest that KIT plays a predominant role in normal erythropoiesis but may be important in regulating some types of malignant hematopoietic cell growth as well. They also suggest that KIT expression is linked to cell metabolic activity and that its expression may be regulated by or coregulated with MYB.

Generation of a 50,000-member human DNA library with an average DNA insert size of 75-100 kbp in a bacteriophage P1 cloning vector.

A bacteriophage P1 cloning system that permits the isolation and amplification of cloned DNA fragments as large as 100 kbp was described previously. We have now utilized a similar system to generate a 50,000-member human DNA library with DNA inserts ranging in size from 75 to 100 kbp. Two major obstacles were overcome in constructing the library. The first concerned the mcrAB restriction system of Escherichia coli, which degrades DNA containing MeC and interferes with the recovery of cloned human DNA inserts. In the P1 cloning system, the effect of the Mcr restriction activity is to decrease recovery of cloned inserts by about 35-fold when the activity is in the host cell line and by about 3-fold when the activity is in the cells used to prepare the packaging extract. To circumvent this problem we inactivated, by mutation, the McrAB proteins in both components of the cloning system. The second obstacle concerned the preferential cloning of small DNA fragments from a population of fragments ranging in size from 20 to 100 kbp. To deal with this problem we first modified the P1 lysogen used to prepare the in vitro head-tail packaging extract so that it would produce 12 times as many large P1 heads (head capacity about 110 kbp) as small P1 heads (head capacity about 45 kbp). We then restructured the P1 cloning vector so that it could be used to produce vector "arm" fragments that could be ligated to insert DNA at only one end. This prevented the formation of long concatamers consisting of alternating units of vector and insert DNA and prohibited the packaging of small inserts in large phage heads. Using the insert-biased large head extract, the arms vector, and size-selected human DNA fragments, we showed that as much as 90% of recovered transformants contained inserts in the desired high molecular weight range.