Peter J. Hahn

X-ray induction of methotrexate resistance due todhfr gene amplification

The effect of ionizing radiation on methotrexate (MTX) resistance and gene amplification in cultured mammalian cells was investigated. X-irradiation of mouse EMT-6 cells induced cell killing and MTX resistance due to amplification of the dihydrofolate reductase (dhfr) gene in a dose-dependent manner. The highest yields of mutant cells were obtained at approximately D37 (the dose at which 37% of the cells survive), where the frequency of MTX-resistant cells was four- to eightfold over that of the unirradiated population. The proportion of MTX-resistant cells among the survivors increased logarithmically with dose, up to a 1000-fold increase over unirradiated cells at 1000 cGy, the highest dose tested. The induced frequency of MTX resistance after X-irradiation was greater than the induced frequency of 8-azaguanine resistance, which indicates deletion of the hypoxanthine phosphoribosyltransferase gene. Inhibition of poly(ADP-ribose) polymerase by the addition of 3-aminobenzamide before irradiation increased both cell killing and MTX resistance. Metaphase spreads of chromosomes from EMT-6 cells that had been irradiated and subjected to stepwise increases in MTX concentration showed numerous double minutes. Pulsed-field gel electrophoresis of the DNA from cells containing radiation-induced double minutes showed that many copies of the dhfr gene were present on circular DNA molecules of 106, 2× 106, and 3×106 base pairs. These results suggest a relationship between the induction of chromosome aberrations and the induction of gene amplification.

Hyperthermia inhibits the repair of DNA double-strand breaks induced by ionizing radiation as determined by pulsed-field gel electrophoresis

Hyperthermia is known to synergistically interact with X-rays to kill cells. We have used pulsed-field gel electrophoresis to investigate the effects of hyperthermia on cell survival and on repair of radiation-induced DNA double-strand breaks (dsbs). Combining hyperthermia (43 degrees C, 45 min) with radiation (7.5 Gy) resulted in a complete inhibition of dsb repair and a surviving fraction of 0.9%. Cells treated with hyperthermia alone resulted in a 55% cell survival with no increase in dsb levels over background. Cells treated with 7.5 Gy alone demonstrated 11% survival and exponential dsb repair. Dsb repair was equally inhibited by hyperthermia whether administered immediately before or after the radiation. We compared the rejoining of dsbs resulting from 7.5 Gy at 37 and 43 degrees C to determine whether dsbs were being repaired during hyperthermia. While repair occurred at 37 degrees C, no dsbs were repaired at 43 degrees C. Our results indicate that hyperthermia completely inhibits dsb repair.

Hydroxyurea Accelerates the Loss of Epidermal Growth Factor? Receptor Genes Amplified As Double-minute Chromosomes in Human Glioblastoma Multiforme

OBJECTIVE We sought to determine whether hydroxyurea could accelerate the loss of amplified epidermal growth factor receptor (EGFR) genes from glioblastoma multiforme (GBM). There is good reason to think that elimination of amplified EGFR genes from GBMs will negatively impact tumor growth. Hydroxyurea has previously been shown to induce the loss of amplified genes from extrachromosomal double minutes (dmin) but not from chromosomal homogeneously staining regions. METHODS Pulsed-field gel electrophoresis and Southern blot hybridization were used to demonstrate EGFR genes amplified as dmin. Giemsa-stained metaphase spreads were prepared in an attempt to visualize dmin. A GBM cell line containing amplified EGFR genes was treated continuously in vitro with 0 to 150 mumol/L hydroxyurea, and slot blot analysis was used to show the loss of amplified EGFR genes. RESULTS Amplified EGFR genes were found on dmin in 4 of 11 (36%) fresh human GBM biopsy specimens. None of the GBMs contained EGFR genes amplified as homogeneously staining regions. Amplified dmin were not microscopically visible when stained with Giemsa because of their small size. Slot blot analysis showed that these low doses of hydroxyurea accelerated the loss of amplified EGFR genes in a dose- and time-dependent fashion. Pulsed-field gel electrophoresis and Southern blot analysis confirmed that EGFR gene loss was accompanied by amplified dmin loss in a dose-dependent fashion. CONCLUSION These studies suggest the potential use of low-dose hydroxyurea in the treatment of GBMs.

Molecular structure and evolution of double-minute chromosomes in methotrexate-resistant cultured mouse cells.

To determine whether microscopically visible double-minute chromosomes (DMs) are derived from submicroscopic precursors, we monitored the amplification of the dihydrofolate reductase (DHFR) gene in 10 independent isolates of methotrexate (MTX)-resistant mouse cells. At every other doubling in MTX concentration, the cells were examined both microscopically, to detect the presence of microscopically visible DMs, and by pulsed-field gel electrophoresis and hybridization to a DHFR-specific probe, to detect submicroscopic DMs. One of the cloned MTX-resistant isolates was examined in detail and was shown to originally contain amplified DHFR genes on circular DMs measuring 1 and 3 Mb in size; additionally, metaphase chromosome preparations from this cloned isolate were examined and were shown to contain microscopically visible DMs too large to enter a pulsed-field gel. During stepwise selection for increasing levels of MTX, the smaller DMs (not microscopically visible) were shown to be preferentially amplified, whereas the larger (microscopically visible) ones decreased in relative numbers. Rare-cutting NotI digestion patterns of total genomic DNA that includes the DMs containing the DHFR gene suggest that the DMs increase in copy number without any further significant rearrangements. We saw no evidence from any of the 10 isolates to suggest that microscopically visible DMs are formed from smaller submicroscopic precursors.

Induction and repair of DNA double-strand breaks in the same dose range as the shoulder of the survival curve

We have used pulsed-field gel electrophoresis (PFGE) to test two hypotheses that have been proposed to explain the survival curves with shoulders which are characteristic of low-LET ionizing radiation: (1) Neutral elution studies of the induction of double-strand breaks (DSBs) have suggested that ionizing radiation might induce DSBs in a nonlinear fashion at low doses. (2) Based on analogies to enzyme kinetics, DSB repair might be saturating in the shoulder region. We quantified DSB induction and survival resulting from doses between 0 and 5 Gy spanning the shoulder region of the survival curve. We found that DSB induction was linear at all doses tested down to 0.5 Gy, the limits of sensitivity. Therefore, nonlinear DSB induction cannot account for the shape of the survival curve. To determine whether the DSB repair system was saturated in the shoulder region, we quantified the rate of DSB repair as a function of dose of X rays between 1.25 and 20 Gy. The repair of DSBs was exponential with half-times of repair constant for doses below 10 Gy, and averaged 28 min. We determined the initial rate of repair from the exponential repair kinetics for each dose. The initial rate of repair after radiation treatment increased linearly with dose up to at least 10 Gy. Therefore, saturating DSB repair cannot explain the shoulder of the survival curve.

CpG island mapping of a mouse double-minute chromosome.

The development of double-minute chromosomes (DMs) and subsequent gene amplification are important genomic alterations resulting in increased oncogene expression in a variety of tumors. The molecular mechanisms mediating the development of these acentric extrachromosomal elements have not been completely defined. To elucidate the mechanisms involved in DM formation, we have developed strategies to map amplified circular DM DNA. In this study, we present a long-range restriction map of a 980-kb DM. A cell line cloned from mouse EMT-6 cells was developed by stepwise selection for resistance to methotrexate. This cloned cell line contains multiple copies of the 980-kb DM carrying the dihydrofolate reductase (DHFR) gene. A long-range restriction map was developed in which a hypomethylated CpG-rich region near the DHFR gene served as a landmark. This strategy was combined with plasmid-like analysis of ethidium bromide-stained pulsed-field gels and indicated that a single copy of the DHFR gene was located near a hypomethylated region containing SsII and NotI sites. At least 490 kb of this DM appears to be composed of unrearranged chromosomal DNA.

Differential sensitivity of double minute chromosomes to hydroxyurea treatment in cultured methotrexate-resistant mouse cells

Treating mammalian cells with continuous sub-lethal doses of Hydroxyurea (HU) causes the loss of double minute chromosomes (DMs) containing amplified oncogenes in culture. Recently, we have shown that treating glioblastoma multiforme cells in culture with low doses of HU causes the loss of DMs containing epidermal growth factor receptor genes. Loss of amplified EGFR genes was accompanied by cessation of growth, and greatly decreased tumorigenicity. To further study HU-induced elimination of DMs we have now followed the fate of dihydrofolate reductase gene (DHFR) amplifying DMs in methotrexate-resistant mouse cells during simultaneous treatment with both MTX and HU. We report that in the presence of both HU and MTX, the amplified genes decreased to 25% of starting levels in the first week of treatment, but that ultimately the cells become resistant to HU and reamplify the DHFR gene. We also report that some DHFR amplifying DMs are much more sensitive to HU than others. This study demonstrates that HU does not simply increase the rate of passive loss of DMs.

Double-minute chromosomes as megabase cloning vehicles

Radiation-reduced chromosomes provide valuable reagents for cloning and mapping genes, but they require multiple rounds of x-ray deletion mutagenesis to excise unwanted chromosomal DNA while maintaining physical attachment of the desired DNA to functional host centromere and telomere sequences. This requirement for chromosomal rearrangements can result in undesirable x-ray induced chromosome chimeras where multiple non-contiguous chromosomal fragments are fused. We have developed a cloning system for maintaining large donor subchromosomal fragments of mammalian DNA in the megabase size range as acentric chromosome fragments (double-minutes) in cultured mouse cells. This strategy relies on randomly inserted selectable markers for donor fragment maintenance. As a test case, we have cloned random segments of Chinese hamster ovary (CHO) chromosomal DNA in mouse EMT-6 cells. This was done by cotransfecting plasmids pZIPNeo and pSV2dhfr into DHFR-CHO cells followed by isolation of a Neo + DHFR + CHO donor colony and radiation-fusion-hybridization (RFH) to EMT-6 cells. We then selected for initial resistance to G418 and then to increasing levels of methotrexate (MTX). Southern analysis of pulsed-field gel electrophoresis of rare-cutting restriction endonuclease digestions of DNA from five RFH isolates indicated that all five contain at least 600 kb of unrearranged CHO DNA. In situ hybridization with the plasmids pZIPNeo and pSV2dhfr to metaphase chromosomes of MTX-resistant hybrid EMT-6 lines indicated that these markers reside on double-minute chromosomes.

Chromosome breakpoints near CpG islands in double minutes

Double minute chromosomes (DMs) are the principal genetic vehicles for amplifying oncogenes in human tumors and drug resistance genes in cultured mouse cells. Mouse EMT-6 cells resistant to methotrexate (MTX) generally contain circular DMs, approximately 1 megabase (Mb) in size, that amplify the dihydrofolate reductase (DHFR) gene. The 1 Mb DMs generally have CpG islands located 500 kb upstream of the DHFR gene. The purpose of this study was to determine the relationship between CpG islands and chromosomal breakpoints giving rise to the DM. We show that EMT-6 cells growing in very low levels of MTX that do not yet contain the 1 Mb DHFR-amplifying DM, develop a NotI/EagI site 500 kb upstream of the DHFR gene. This NotI site is close to, if not identical with, one of the chromosomal breakpoints giving rise to the DM. We show that 500 kb of DM DNA from upstream of the DHFR gene is derived from 500 kb of chromosomal DNA upstream of the chromosomal DHFR gene. The downstream breakpoint maps to a region approximately 200 kb downstream of the DHFR gene near a chromosomal SstII/EagI site. Therefore, approximately 700 kb of DM DNA was derived from the genomic region surrounding the DHFR gene. To confirm the organization of the DM DNA, we isolated DNA probes from the 1 Mb DM. Using pulsed field gel electrophoresis and Southern hybridization, we determined the approximate location of each probe with respect to the CpG island in both the DM and the chromosome. Approximately 300 kb of chimeric DNA from a region unrelated to the DHFR gene was incorporated during DM formation. Implications for the mechanism of DM formation are discussed.

Variation in genomic Alu repeat density as a basis for rapid construction of low resolution physical maps of human chromosomes

Human DNA restriction fragments containing high numbers of Alu repeat sequences can be preferentially detected in the presence of other human DNA restriction fragments in DNA from human:rodent somatic cell hybrids when the DNA is fragmented with enzymes that cleave mammalian DNA infrequently. This ability to lower the observed human DNA complexity allowed us to develop an approach to order rapidly somatic hybrid cell lines retaining overlapping human genomic domains. The ordering process also generates a relative physical map of the human fragments detected with Alu probe DNA. This process can generate physical mapping information for human genomic domains as large as an entire chromosome (100,000 kb). The strategy is demonstrated by ordering Alu-detected NotI fragments in a panel of mouse:human hybrid cells that span the entire long arm of human chromosome 17.