HIV clinical trials in correctional settings: right or retrogression?

The right of incarcerated prison and jail inmates to health care is protected by the 8th and the 14th amendments of the Constitution, respectively. Does the right to health care include access to clinical trials? At the time of this writing, clinical trials have become part of the fabric of HIV/AIDS care, allowing patients to participate in studies of new and often lifesaving treatments. Participation in trials can also be dangerous, as illustrated by the recent death of a subject in a gene therapy trial. This danger is compounded by ethical dilemmas that can arise from the large amount of financial support for clinical trials (greater than 75%) that is derived from for-profit corporations. Indeed, clinical trials are the subject of grave concern on the part of the United States Government, which has recently taken steps to shore up human subject safeguards. Following a conference on the conduct of clinical trials in correctional settings, the Office for Human Research Protections suspended prison research conducted by 4 prestigious academic institutions.

Analysis of the functional role of chromosome 10 loss in human glioblastomas.

Molecular and cytogenetic analyses of primary brain tumors have shown that losses on chromosome 10 occur very frequently in human glioblastoma multiforme suggesting the presence of a glioma-associated tumor suppressor gene on this chromosome. To examine this hypothesis, a copy of chromosome 10 derived from a human fibroblast cell line was introduced into the human glioma cell line U251 by microcell-mediated chromosomal transfer. A human chromosome 2 was also independently introduced into U251 cells. The presence of novel chromosomes or chromosomal fragments was confirmed by molecular and karyotypic analyses. The hybrid clones containing a transferred chromosome 10 exhibited a suppression of their transformed and tumorigenic phenotype in vivo and in vitro, whereas cells containing a transferred chromosome 2 failed to alter their phenotype. The hybrid cells containing a transferred chromosome 10 displayed a significant decrease in their saturation density and an altered cellular morphology at high cell density but only a slight decrease in their exponential growth rate. A dramatic decrease was observed in the ability of cells with an introduced chromosome 10 to grow in soft agarose. The introduction of chromosome 10 completely suppressed tumor formation when the hybrid cells were injected into nude mice. These findings indicate that chromosome 10 harbors a tumor suppressor gene that is directly involved in glioma oncogenesis.

Direct formation of microcells from mitotic cells for use in chromosome transfer.

Microcells, cytoplasmic fragments that contain micronuclei composed of one or a few chromosomes, can be generated directly from mitotic cells. Cytochalasin B, which causes nuclear extrusion in interphase cells, has a similar effect on the chromosomes of colcemid-blocked mitotic cells. The forces generated during centrifugation in a Percoll gradient are sufficient to separate the extruded microcells from the parent cell. The chromosomes contained in an extruded microcell form micronuclei during the process, and in all respects are comparable to microcells generated from micronucleated cells except that they are uniformly in the G1 phase of the cell replication cycle. The procedure is probably applicable to all mammalian cells that grow in culture and can be employed to make microcells for the transfer of both intact and fragmented chromosomes.

Human oncogenes in mouse/human microcell hybrids.

DNA-mediated gene transfer has been used successfully in many experiments to identify and isolate transforming sequences from human tumors and tumor cell lines. This work was done to compare the efficiency of that technique to microcell-mediated chromosome transfer in the transformation of NIH 3T3 mouse fibroblast cells. Our hope was that oncogenes introduced into a cell in chromosomal form would also be effective in the transformation of NIH 3T3 cells. The study revealed, however, that microcells from cell lines that contain transforming sequences identified by DNA-mediated gene transfer were unable to transform NIH 3T3 cells, although other human genes were expressed in the microcell hybrids. Several possible mechanisms are given and discussed. This research may provide important insight into the possible control of oncogenes in intact human tumor cells.

A general survey of genetics and cancer.

As the molecular details of cancer have begun to unfold, it has become apparent that the cellular genetic apparatus is defective in the malignant cell. Accordingly, we have attempted to review the prominent aspects of genetics research as it impinges on the problem of cancer. Although the field is immense, we have tried to cover four major areas: human genetics, molecular genetics, somatic cell genetics, and developmental genetics. Oncogenes are considered in detail in all of these areas, and a new map (42 entries) of oncogene positions on human chromosomes is presented.

An aberrant avian leukosis virus provirus inserted downstream from the chicken c-myc coding sequence in a bursal lymphoma results from intrachromosomal recombination between two proviruses and deletion of cellular DNA.

A chicken bursal lymphoma, LL6, contains avian leukosis virus DNA integrated 3' of the c-myc coding sequences, unlike all other examined bursal lymphomas, which have integrations 5' to c-myc. To better understand this unusual mutation, we examined a molecular clone containing the LL6 c-myc gene and determined the structure of the proviral insertion by DNA sequencing. Viral DNA begins 575 base pairs downstream of the c-myc coding sequences within the untranslated region, disrupting the use of the normal polyadenylation signal. An internal deletion of the provirus extends from within U3 in the 5' long terminal repeat to within the gp37-coding region of the env gene, disabling virus replication and protein synthesis. Both host-virus boundaries appear normal with respect to the site in viral DNA which is joined to host DNA; both long terminal repeats lack the terminal dinucleotide found in unintegrated DNA. However, in contrast to normal integrations, the six bases of cellular sequence at the 5' junction are not repeated at the 3' junction. The DNA sequences immediately downstream of the LL6 recombinant provirus are not part of the c-myc gene; they originate from the same chromosome as c-myc, but at least 15 kilobases (kb) away. In addition, DNA sequences normally residing 3' of c-myc are deleted in LL6. In summary, these results imply that the LL6 provirus is the result of recombination between two proviruses; that both proviruses were originally downstream of c-myc in the same orientation and separated by at least 15 kb; and that the recombination event was preceded, accompanied, or followed by an internal proviral deletion. No transcript could be detected within a 20-kb region downstream of the LL6 provirus, leaving unresolved the question of whether the additional chromosomal alterations make a specific contribution to LL6 tumorigenesis.

General protocol for microcell-mediated chromosome transfer.

We have developed a general technique for making micronucleated cells to use in microcell-mediated chromosome transfer. Growing cells are blocked in mitosis with colcemid, placed in a hypotonic solution for 10 min, and returned to culture medium for 24 h. This treatment promotes the formation of micronuclei within lymphoblast or fibroblast cells. The microcells are generated by cytochalasin B treatment on a Percoll density gradient centrifuged at 43,500g. The resulting mixture of microcells, whole cells, and karyoplasts is filtered through 3-micron pores to obtain a pure microcell preparation. The microcells are fused to recipient whole cells using phytohemagglutinin-P and polyethylene glycol. Advantages of this technique are: donor cells need not be attached to a substrate; and cell lines which form micronuclei in low frequency can still be used efficiently as microcell donors.

Dispersed chromosomal localization of the proto-oncogenes transduced into the genome of Mill Hill 2 or E26 leukemia virus.

Both Mill Hill 2 and E26 retroviruses have transduced two cellular genes--c-myc and c-mil/mht (Mill Hill 2) and c-myb and c-ets (E26). We localized the genes transduced by these viruses to different chromosomes: c-myc and c-myb to relatively large chromosomes and c-mil/mht and c-ets to microchromosomes. Thus, like avian erythroblastosis virus, each of these retroviruses has transduced two cellular genes unlinked in the chicken genome.

A theory for developmental control by a program encoded in the genome.

A new genetic mechanism is proposed to explain the evident order seen in embryonic development. This theory postulates control DNA, a set of genetic elements activated in a specific sequence, one at a time. With each cell division, control of gene expression passes to the next control unit in the series. The complete series of control units would constitute the encoded (and inherited) development program of an organism.

Cellular oncogenes (c-erb-A and c-erb-B) located on different chicken chromosomes can be transduced into the same retroviral genome.

Avian erythroblastosis virus has transduced two cellular genes, c-erb-A and c-erb-B. Using fractionated chicken chromosomes, we found that the two genes are located on different chromosomes in the chicken genome: c-erb-A is on a microchromosome, and c-erb-B is on a large chromosome. The locations of two other cellular oncogenes (c-fps and c-myb) were also determined: c-fps is on a microchromosome, and c-myb is on chromosome of an intermediate size. Our results suggest that avian erythroblastosis virus had transduced the two cellular genes independently, conforming to previous indications that cellular oncogenes are dispersed among multiple chromosomes in every species that has been examined.

Recombinant DNA strategies in genetic neurological diseases.

The application of recombinant DNA techniques applied to the study of genetic neurological diseases will play a major role in the practice of neurology in upcoming years. Strategies are now available to develop useful and relatively simple biochemical diagnostic tests for heterozygous individuals with diseases inherited as autosomal dominant traits. In addition, molecular genetic methods will lead to the delineation of the genomic mutations responsible for these diseases. This review will update the current status of research in several neurological genetic diseases including myotonic muscular dystrophy, Huntington's disease, Charcot-Marie-Tooth disease and Duchenne muscular dystrophy (X-linked). An introduction and overview of the methodology is provided. Specific research strategies including random screening of libraries, chromosome walking, messenger RNA selection, and messenger RNA translation are described. These strategies are designed to provide heterozygote identification, prenatal diagnosis and gestational management, the development of rational therapies, and the understanding of the molecular basis of disease expression.

Multiple alpha and beta tubulin genes represent unlinked and dispersed gene families.

Cloned cDNA sequences specific for alpha or beta tubulin mRNAs have been used to show that the multigene families which encode either alpha or beta tubulin are unlinked and dispersed throughout the chicken genome. Fractions of chicken chromosomes partially purified by centrifugation on a sucrose gradient were digested with restriction endonucleases and electrophoresed on agarose gels. The DNA was transferred to nitrocellulose filters and hybridized to labeled probes constructed from cloned cDNA sequences specific for alpha or beta tubulin. We find alpha tubulin sequences on four different chicken chromosomes and beta tubulin sequences on at least two different chromosomes. Moreover, using chicken chromosomes further purified with a fluorescent cell sorter, we have been able unambiguously to localize alpha tubulin genes to chromosome 1 and chromosome 8 and two of the beta genes to chromosome 2.

Chromosome bands and the subunit structure of Chinese hamster metaphase chromosomes.

Isolated Chinese hamster chromosomes dissociate into a series of specific chromatin subunits approximately the size of stainable chromosome bands upon reduction of the divalent ion concentration during or after isolation. At high pH the chromatin in some bands is differentially removable during chromosome isolation, leaving a banded chromosome with a pattern typical of most G-band procedures. This provides an alternate molecular mechanism to explain the production of banded chromosomes by a variety of staining procedures. These results also suggest an approach to chromatin fractionation, using metaphase chromosomes as a starting material.