Continuation of mitosis after selective laser microbeam destruction of the centriolar region.

The centriole regions of prophase PTK2 cells were irradiated with a laser microbeam. Cells continued through mitosis normally. Ultrastructural analysis revealed either an absence of centrioles or severely damaged centrioles at the irradiated poles. Microtubules appeared to focus into pericentriolar cloud material.

Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfecti): new evidence from ultrastructural and laser microbeam experiments.

The anaphase-telophase spindle usually elongates, and it has been assumed that the spindle pushes the incipient daughter nuclei apart. To test this assumption, we used a laser microbeam to sever the central spindle of the fungus, Fusarium solani, and measured the rate of separation of incipient daughter nuclei. When the microbeam was aimed beside the spindle separation occurred at a rate (8.6 micrometer/min) that did not differ significantly from the rate (7.6 micrometer/m) in unirradiated cells. But when the spindle was irradiated, it broke, and the separation was much faster (22.4 micrometer/min). Irradiation of cytoplasm lateral to one spindle pole resulted in a 1.5 micrometer/min reduction in the rate (6.1 micrometer/min) of separation. From these and other data, we infer that extranuclear forces, presumably involving astral microtubules, pull on the incipient daughter nuclei and that the central spindle limits the separation rate. Astral microtubules are associated with the plasma membrane or, sometimes, with the rough endoplasmic reticulum. Most of the spindle microtubules that are present at metaphase are depolymerized during anaphase and early telophase.

Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells.

The role of the kinetochore in chromosome movement was studied by 532-nm wavelength laser microirradiation of mitotic PtK2 cells. When the kinetochore of a single chromatid is irradiated at mitotic prometaphase or metaphase, the whole chromosome moves towards the pole to which the unirradiated kinetochore is oriented, while the remaining chromosomes congregate on the metaphase plate. The chromatids of the irradiated chromosome remain attached to one another until anaphase, at which time they separate by a distance of 1 or 2 micrometers and remain parallel to each other, not undergoing any poleward separation. Electron microscopy shows that irradiated chromatids exhibit either no recognizable kinetochore structure or a typical inactive kinetochore in which the tri-layer structure is present but has no microtubules associated with it. Graphical analysis of the movement of the irradiated chromosome shows that the chromosome moves to the pole rapidly with a velocity of approximately 3 micrometers/min. If the chromosome is close to one pole at irradiation, and the kinetochore oriented towards that pole is irradiated, the chromosome moves across the spindle to the opposite pole. The chromosome is slowed down as it traverses the equatorial region, but the velocity in both half-spindles is approximately the same as the anaphase velocity of a single chromatid. Thus a single kinetochore moves twice the normal mass of chromatin (two chromatids) at the same velocity with which it moves a single chromatid, showing that the velocity with which a kinetochore moves is independent, within limits, of the mass associated with it.

Laser-transected microtubules exhibit individuality of regrowth, however most free new ends of the microtubules are stable.

To study the possible mechanism of microtubule turnover in interphase cells, we have used the 266-nm wavelength of a short-pulsed Nd/YAG laser to transect microtubules in situ in PtK2 cells at predefined regions. The regrowth and shrinkage of the transected microtubules have been examined by staining the treated cells with antitubulin mAb at various time points after laser irradiation. The results demonstrate that microtubules grow back into the transected zones individually; neither simultaneous growth nor shrinkage of all microtubules has been observed. The half-time of replacement of laser-dissociated microtubules is observed to be approximately 10 min. On the other hand, exposure of the core of the microtubule, which is expected to consist almost completely of GDP-tubulin, by transecting the internal regions of the microtubule does not render the remaining polymer catastrophically disassembled, and most transected microtubules with free minus ends do not quickly disappear. Taken together, these results suggest that most microtubules in cultured interphase cells exhibit some properties of dynamic instability (individual regrowth or shrinkage); however, other factors in addition to the hydrolysis of GTP-tubulin need to be involved in modulating the dynamics and the stability of these cytoplasmic microtubules.

The role of the centriolar region in animal cell mitosis. A laser microbeam study.

An argon ion laser microbeam (488 and 514 nm) was used to selectively irradiate one of the two centriolar regions of rat kangaroo Potorous tridactylis (PtK2) prophase cells in vitro. The cells were sensitized to the laser radiation by treatment with acridine orange (0.1-0.2 mug/ml). Ultrastructural examination of the irradiated centriolar regions demonstrated that the primary site of damage was the pericentriolar material. This result suggests that nucleic acid is present in the pericentriolar material. Behavioral and ultrastructural analysis demonstrated that cells with one damaged pericentriolar zone could undergo (a) nuclear membrane breakdown, (b) chromosome condensation, (c) metaphase plate formation, and (d) cytokinesis. However, the chromosomes neither separated nor exhibited any anaphase movements. Detailed ultrastructural analysis revealed the presence of kinetochore microtubules on both sides of chromosome mass and a lack of microtubules in the cytokinesis constriction. These results indicate that the pericentriolar material is important in spindle orgainization and essential for the formation of the interpolar microtubules.

The absence of centrioles from spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division in vitro.

Light and electron microscopy were used to study somatic cell reduction division occurring spontaneously in tetraploid populations of rat kangaroo Potorous tridactylis (PtK2) cells in vitro. Light microscopy coupled with time-lapse photography documented the pattern of reduction division which includes an anaphase-like movement of double chromatid chromosomes to opposite spindle poles followed by the organization of two separate metaphase plates and synchronous anaphase division to form four poles and four daughter nuclei. The resulting daughter cells were isolated and cloned, showing their viability, and karyotyped to determine their ploidy. Ultrastructural analysis of cells undergoing reduction consistently revealed two duplexes of centrioles (one at each of two spindle poles) and two spindle poles in each cell that lacked centrioles but with microtubules terminating in a pericentriolar-like cloud of material. These results suggest that the centriole is not essential for spindle pole formation and division and implicate the could region as a necessary component of the spindle apparatus.

Laser irradiation of centrosomes in newt eosinophils: evidence of centriole role in motility.

Newt eosinophils are motile granulated leukocytes that uniquely display a highly visible centrosomal area. Electron microscope and tubulin antibody fluorescence confirms the presence of centrioles, pericentriolar material, and radiating microtubules within this visible area. Actin antibodies intensely stain the advancing cell edges and tail but only weakly stain pseudopods being withdrawn into the cell. Randomly activated eosinophils follow a roughly consistent direction with an average rate of 22.5 micron/min. The position of the centrosome is always located between the trailing cell nucleus and advancing cell edge. If the cell extends more than one pseudopod, the one closest to or containing the centrosome is always the one in which motility continues. Laser irradiation of the visible centrosomal area resulted in rapid cell rounding. After several minutes following irradiation, most cells flattened and movement continued. However, postirradiation motility was uncoordinated and directionless , and the rate decreased to an average of 14.5 micron/min. Electron microscopy and tubulin immunofluorescence indicated that an initial disorganization of microtubules resulted from the laser microirradiations . After several minutes, organized microtubules reappeared, but the centrioles appeared increasingly damaged. The irregularities in motility due to irradiation are probably related to the damaged centrioles. The results presented in this paper suggest that the centrosome is an important structure in controlling the rate and direction of newt eosinophil motility.

Studies of a microtubule-associated protein using a monoclonal antibody elicited against mammalian mitotic spindles.

We have devised a procedure for the identification of individual molecules which are associated with the mitotic spindle apparatus and cytoskeleton in mammalian cells. We prepared monoclonal antibody-producing hybridomas by immunizing mice with mitotic spindles isolated from cultured HeLa cells. Among several antibody-producing clones obtained, one hybridoma (22MA2) produced an antibody that recognizes a putative microtubule-associated protein which exhibits unusual distribution characteristics in cultured cells. Immunofluorescence studies showed that during mitosis the 22MA2 antigen is distributed in parallel with the spindle fibers of the mitotic apparatus, and that during interphase the antigen is always associated to a limited extent with cytoplasmic microtubules. Also, the co-distribution of the antigen with microtubules was found to be Colcemid sensitive. However, the 22MA2 antibody immunofluorescently stained the nuclei of cells in the exponential growth phase, but did not stain the nuclei of cells that had grown to confluence. This nuclear fluorescence appears to be directly related to cell density rather than nutritional (serum) factors in the growth medium. The results suggest that the antigen undergoes some change in structure or distribution in response to changes in the proliferative capacity of the cell. Biochemical analyses of cytoplasmic, nuclear, and mitotic spindle subcellular fractions show that the antigen exhibits a polypeptide molecular weight of 240,000 is found in various mammalian cells ranging from marsupial to human, and is particularly susceptible to proteolysis.

Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients.

The specificity of the staining of CREST scleroderma patient serum was investigated by immunofluorescence and immunoelectron microscopy. The serum was found to stain the centromere region of mitotic chromosomes in many mammalian cell types by immunofluorescence. It also localized discrete spots in interphase nuclei which we have termed "presumptive kinetochores." The number of presumptive kinetochores per cell corresponds to the chromosome number in the cell lines observed. Use of the immunoperoxidase technique to localize the antisera on PtK2 cells at the electron microscopic level revealed the specificity of the sera for the trilaminar kinetochore disks on metaphase and anaphase chromosomes. Presumptive kinetochores in the interphase nuclei were also visible in the electron microscope as randomly arranged, darkly stained spheres averaging 0.22 micrometers in diameter. Preabsorption of the antisera was attended using microtubule protein, purified tubulin, actin, and microtubule-associated proteins. None of these proteins diminished the immunofluorescence staining of the sera, indicating that the antibody-specific antigen(s) is a previously unrecognized component of the kinetochore region. In some interphase cells observed by both immunofluorescence and immunoelectron microscopy, the presumptive kinetochores appeared as double rather than single spots. Analysis of results obtained using a microspectrophotometer to quantify DNA in individual cells double stained with scleroderma serum and the DNA fluorescent dye, propidium iodide, led to the conclusion that the presumptive kinetochores duplicate in G2 of the cell cycle.

Directed chromosome loss by laser microirradiation.

In this article I have presented data that indicate the feasibility of attaining the five objectives outlined in the introduction. It should be possible to assign genes to specific chromosome regions by (i) selective DNA deletion of a 0.25- to 0.5-micro.m segment of one or both homologous chromosomes, (ii) deletion of one or both entire homologous chromosomes, or (iii) combining cell fusion with selective deletion of whole chromosomes and then deletion of chromosome segments. By laser microirradiation it should be possible to determine which chromosomes and chromosome regions are essential for immediate cell survival by removing from individual cells whole chromosomes, and chromosome segments from each of the chromosomes in the karyotype, and then assessing the cloning efficiency of each cell. For example, we have already determined that removal of one large chromosome No. 1 from PTK(2) cells does not prevent the cell from undergoing a subsequent mitosis. It should also be possible to generate new classes of mutants by damaging small selected areas of DNA with the laser beam and then cloning the irradiated cells-but this has yet to be demonstrated. This procedure might reveal recessive alleles on the nonirradiated homolog, or might result in the direct production of a genetic mutation. Irradiation of identical places on both homologous chromosomes could result in deletion of a genetic locus which ultimately might be detected as a deficiency in a metabolic pathway or some other cellular abnormality. Studies on chromosome stability and DNA constancy can be conducted with laser irradiated cells. For example, the karyotypic analysis of chromosome No. 1 suggests that a cellular mechanism exists to maintain the constancy of this chromosome in both the diploid and tetraploid cell lines. The same approach could be used with each of the chromosomes in the karyotype. Various cytochemical procedures could be used for making quantitative DNA studies of the cells, and chromosome and DNA analyses could be performed at varying times following laser microirradiation. It might also be possible to study the repair of chromosomal damage caused by laser irradiation. The cells could be examined by autoradiographic, cytochemical, and electron microscopy procedures at varying times after irradiation, and because the precise location, time, and nature of the mutational event would be known, subsequent analysis of repair and alteration would be facilitated.

Cloning of rat kangaroo (PTK2) cells following laser microirradiation of selected mitotic chromosomes.

Mitotic chromosomes of rat kangaroo cells were irradiated with a green argon laser microbeam without prior dye sensitization. Deoxyribonucleic acid-negative chromosome paling was observed. The irradiated cells were isolated and cloned into viable populations.

Regulation of segment-building during the postembryonic development of a common milliped.

Starvation of larvae of the milliped Narceus annularis results in the formation of a smaller than normal number of body segments in the early stadia. However, this reduced number is compensated for by subsequent formation of a larger than normal number of body segments in the later stadia, or by the addition of extra segment-building stadia. Apparently there is a mechanism whereby the milliped "keeps count" of the number of body segments produced and can regulate the production of these segments from a proliferative region so that the total number of body segments at sexual maturity is within the normal range for the species.

Chromosome lesions produced with an argon laser microbeam without dye sensitization.

Improvements in the argon laser microbeam have made it possible to cause damage to chromosomes of tissue culture cells without prior treatment of the cells with a photosensitizing agent. These results have been confirmed independently in two laboratories.

Enzyme inactivation with ultraviolet laser energy (2650 Angstroms).

Inactivation of rat heart lactate dehydrogenase was accomplished by irradiation of the enzyme in solution with a frequency quadrupled neodymium glass laser.

Laser microsurgery in cell and developmental biology.

New applications of laser microbeam irradiation to cell and developmental biology include a new instrument with a tunable wavelength (217- to 800-nanometer) laser microbeam and a wide range of energies and exposure durations (down to 25 X 10(-12) second). Laser microbeams can be used for microirradiation of selected nucleolar genetic regions and for laser microdissection of mitotic and cytoplasmic organelles. They are also used to disrupt the developing neurosensory appendages of the cricket and the imaginal discs of Drosophila.

Comparison of fluorescence and photodynamic activities of whole hematoporphyrin derivative and its enriched active components.

The in vivo biologic activities of the hematoporphyrin derivative (Photofrin) and the enriched, so-called "active fraction" (Photofrin II) were determined by measuring the necrosis produced in implanted tumors in DBA/2Ha mice exposed to various total doses of light (20-100 J/cm2) after ip administration of 10 mg/kg standard doses of either Photofrin or Photofrin II. Total relative percentage increase in fluorescence in tumor tissue, as compared to fluorescence in control tissue, also was measured for both Photofrin and Photofrin II. In response to total light doses (630 nm) of 40-100 J/cm2, mice that received Photofrin had comparable amounts of tumor necrosis to those mice that received Photofrin II. At doses of 40-60 J/cm2, 80% tumor destruction resulted, and at 80-100 J/cm2, tumor destruction was 100%. However, at a total light dose of 20 J/cm2, the tumors that received Photofrin II exhibited 60-80% tumor necrosis, whereas those animals that received Photofrin had only small areas of patchy necrosis associated with signs of vascular thrombosis and hemorrhage into the surrounding perivascular stroma. A 25.2% total increase in maximal tissue fluorescence over that in controls was observed for animals that received Photofrin II, as compared to 13.9% for those animals that received Photofrin. It is concluded that the greater demonstrable efficacy of treatment with Photofrin II, as compared to treatment with Photofrin, is due to enrichment of those nonpolar hydrophobic components of the hematoporphyrin derivative mixture that are thought to be primarily responsible for the in vivo biologic activities.

Photodynamic therapy of human malignant melanoma xenografts in athymic nude mice.

While photodynamic therapy (PDT) for cutaneous malignancies including dermal recurrences of breast cancer and basal cell carcinomas has shown great promise, PDT of malignant melanoma has remained incompletely understood. A comparison study of the effects of PDT on human xenograft amelanotic and melanotic malignant melanoma in the athymic nude mouse model was performed. Twenty-four hours after ip administration of Photofrin II, the responses to total laser light doses of 25-300 J/cm2 were evaluated by histologic examination. Animals were also sacrificed 24 hours after administration of Photofrin II without light, and their uptake and localization of hematoporphyrin derivative (HpD) for each tumor were measured and compared. The results indicate that human xenograft melanotic melanoma, despite the fact that it contains more HpD than does amelanotic melanoma, is far less responsive to PDT. This result appears to be due to the competing chromophore melanin, which may inhibit the photochemical reaction at several key points.

Photodynamic therapy of spontaneous cancers in felines, canines, and snakes with chloro-aluminum sulfonated phthalocyanine.

This is the first report on the photodynamic treatment with a second-generation sensitizer, chloro-aluminum sulfonated phthalocyanine (CASPc) of spontaneously arising tumors and on the photodynamic therapy (PDT) of snake neoplasms. Each of 10 cats, 2 dogs, and 3 snakes presenting with a variety of tumor types (squamous cell carcinoma, mast cell malignant tumor, and mixed carcinoma/sarcoma) was given an intravenous injection of 1 mg of CASPc per kilogram body weight 48 hours prior to irradiation with 675-nm light. Some larger tumors (greater than 1.5 cm deep) were surgically debulked prior to PDT. No significant systemic toxicity or skin photosensitization was observed in any animal. The tumor responses were comparable to those seen with conventional cryotherapy, hyperthermia, or surgery. PDT with CASPc of these cases led to 67% (12 of 18) complete response, 22% (4 of 18) partial response, and 11% (2 of 18) no response (less than 50% reduction in tumor size). Four cases could not be evaluated. Since the overall tumor response to CASPc is very good, and the problem of skin photosensitivity is nonexistent, it is expected that using CASPc-PDT to eradicate human tumors would also yield comparable results. Further studies with long-term follow-up are necessary to optimize the use of CASPc-PDT in veterinary and human medicine.

In vitro characterization of monoaspartyl chlorin e6 and diaspartyl chlorin e6 for photodynamic therapy.

The characteristics of two new chlorin photosensitizers were studied in cell culture by determining phototoxicity, subcellular localization, and photophysical properties. Monoaspartyl chlorin e6 (MACE) and diaspartyl chlorin e6 (DACE) are new photosensitizers that show promise for use in photodynamic therapy. These chlorins are pure, monomeric compounds as determined by high-pressure liquid chromatography. Both compounds absorb substantially at a longer wavelength (664 nm) than does dihematoporphyrin ether-ester (DHE). Tumor diagnosis with the use of fluorescence should be facilitated due to the purity of the compounds and the single fluorescence emission peak. Phototoxicity dose-response curves of the sensitizers were completed using a standard clonogenic assay to determine cell viability. The chlorins showed good sensitizing capabilities with light. In addition, subcellular localization of MACE, DACE, and DHE was studied using fluorescence microscopy. Whereas DHE was located throughout the cytoplasm, the primary site of localization of the chlorins appeared to be in the lysosome. The results demonstrate that MACE and DACE are effective photosensitizing agents in vitro and compare favorably to DHE.

Mechanism of tumor destruction following photodynamic therapy with hematoporphyrin derivative, chlorin, and phthalocyanine.

The effect of photodynamic therapy on the tumor microvasculature in the first few hours after treatment was studied at the light and electron microscopy levels. BALB/c mice with EMT-6 tumor received ip injections of hematoporphyrin derivative, chlorin, or phthalocyanine, and 24 hours later, the tumors were treated with light at 100 J/cm2 at the appropriate therapeutic wavelength for each photosensitizer. Animals were killed and their tumors removed at time 0, 30 minutes, 1 hour, and 2, 4, 6, 8, 12, 16, and 24 hours after treatment. The results indicate that for all three sensitizers the effects of photodynamic therapy leading to rapid necrosis of tumor tissue are not the result of direct tumor cell kill but are secondary to destruction of the tumor microvasculature. The first observable signs of destruction occur in the subendothelial zone of the tumor capillary wall. This zone, composed of dense collagen fibers and other connective tissue elements, is destroyed in the first few hours after phototherapy. However, the ultrastructural changes seen in this zone are different for the hematoporphyrin derivative, compared with chlorin and phthalocyanine. Binding of photosensitizers to the elements in this zone as well as altered permeability and transport through the endothelial cell layer because of the increased intraluminal pressure may be key features of tumor destruction.