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Radiotherapy constitutes an essential element in the multimodal therapy of Ewing’s sarcoma. Compared to other sarcomas, Ewing tumors normally show a good response to radiotherapy. However, there are consistently tumors with a radioresistant phenotype, and the underlying mechanisms are not known in detail. Here we investigated the association between survivin protein expression and the radiosensitivity of Ewing’s sarcoma in vitro. An siRNA-based knockdown approach was used to investigate the influence of survivin expression on cell proliferation, double-strand break (DSB) induction and repair, apoptosis and colony-forming ability in four Ewing’s sarcoma cell lines with and without irradiation. Survivin protein and mRNA were upregulated in all cell lines tested in a dose-dependent manner. As a result of survivin knockdown, STA-ET-1 cells showed reduced cell proliferation, an increased number of radiation-induced DSBs, and reduced repair. Apoptosis was increased by knockdown alone and increased further in combination with irradiation. Colony formation was significantly reduced by survivin knockdown in combination with irradiation. Survivin is a radiation-inducible protein in Ewing’s sarcoma and its down-regulation sensitizes cells toward irradiation. Survivin knockdown in combination with radiation inhibits cell proliferation, repair, and colony formation significantly and increases apoptosis more than each single treatment alone. This might open new perspectives in the radiation treatment of Ewing’s sarcoma.

Radiotherapy is a powerful cure for several types of solid tumours, but its application is often limited because of severe side effects in individual patients. With the aim to find biomarkers capable of predicting normal tissue side reactions we analysed the radiation responses of cells from individual head and neck tumour and breast cancer patients of different clinical radiosensitivity in a multicentric study. Multiple parameters of cellular radiosensitivity were analysed in coded samples of peripheral blood lymphocytes (PBLs) and derived lymphoblastoid cell lines (LCLs) from 15 clinical radio-hypersensitive tumour patients and compared to age- and sex-matched non-radiosensitive patient controls and 15 lymphoblastoid cell lines from age- and sex- matched healthy controls of the KORA study. Experimental parameters included ionizing radiation (IR)-induced cell death (AnnexinV), induction and repair of DNA strand breaks (Comet assay), induction of yH2AX foci (as a result of DNA double strand breaks), and whole genome expression analyses. Considerable inter-individual differences in IR-induced DNA strand breaks and their repair and/or cell death could be detected in primary and immortalised cells with the applied assays. The group of clinically radiosensitive patients was not unequivocally distinguishable from normal responding patients nor were individual overreacting patients in the test system unambiguously identified by two different laboratories. Thus, the in vitro test systems investigated here seem not to be appropriate for a general prediction of clinical reactions during or after radiotherapy due to the experimental variability compared to the small effect of radiation sensitivity. Genome-wide expression analysis however revealed a set of 67 marker genes which were differentially induced 6 h after in vitro-irradiation in lymphocytes from radio-hypersensitive and non-radiosensitive patients. These results warrant future validation in larger cohorts in order to determine parameters potentially predictive for clinical radiosensitivity.

In recent years, a special type of cancer cell--the cancer stem cell (CSC)--has been identified and characterized for different tumors. CSCs may be responsible for the recurrence of a tumor following a primarily successful therapy and are thought to bear a high metastatic potential. For the development of efficient treatment strategies, the establishment of reliable methods for the identification and effective isolation of CSCs is imperative. Similar to their stem cell counterparts in bone marrow or small intestine, different cluster of differentiation surface antigens have been characterized, thus enabling researchers to identify them within the tumor bulk and to determine their degree of differentiation. In addition, functional properties characteristic of stem cells can be measured. Side population analysis is based on the stem cell-specific activity of certain ATP-binding cassette transporter proteins, which are able to transport fluorescent dyes out of the cells. Furthermore, the stem cell-specific presence of aldehyde dehydrogenase isoform 1 can be used for CSC labeling. However, the flow cytometric analysis of these CSC functional features requires specific technical adjustments. This review focuses on the principles and strategies of the flow cytometric analysis of CSCs and provides an overview of current protocols as well as technical requirements and pitfalls. A special focus is set on side population analysis and analysis of ALDH activity. Flow cytometry-based sorting principles and future flow cytometric applications for CSC analysis are also discussed.

The majority of patients tolerate radiotherapy well, but some of them suffer from severe side effects. To find genes possibly predictive for radiosensitivity, mRNA profiles were generated before and 6 h after in vitro irradiation with 5 Gy. We analyzed lymphocytes from four head and neck and eight breast cancer patients with strong acute radiation toxicity and from 12 matching normal reacting patients in a blind study. Expression was also measured in lymphocyte subpopulations and Epstein–Barr transformed lymphocytes. Radiation response in whole lymphocyte populations was most similar to that of B cells. In peripheral blood lymphocytes of all patients; 153 genes were identified which were statistically significantly altered by a fold change of more than 50% by irradiation. The signatures of radio-responsive genes differed tremendously between primary and transformed cells. Pathway analysis revealed genes involved in p53 signalling, cell cycle control and apoptosis in response to radiation in primary lymphocytes. In these cells, a set of 67 radiationinduced genes was identified capable of differentiating between severe radiosensitive and normal reacting patients. More than one third of such classifying genes belong to the group of apoptosis or cell cycle regulating genes. The classifying potential of the expression signature has now to be validated in further patient cohorts.

To characterize the interaction of tumour necrosis factor alpha (TNF-alpha) and ionising radiation in six sarcoma, lymphoma and carcinoma cell lines.Cells were characterized regarding annexin V/propiduim iodine affinity and caspase-3 status after application of TNF-alpha, radiation, or combined treatment.Three cell lines showed similar results with additive effects of TNF-alpha and radiation in both assays. The other three cell lines significantly differed regarding the detection of apoptotic cells between treatment conditions (radiation and/or TNF application) and between the two apoptosis assays.The interaction between TNF-alpha and radiation differs between tumour entities and cannot be described by using only one parameter.

Background Telomerase activity compensates shortening of telomeres during cell division and enables cancer cells to escape senescent processes. It is also supposed, that telomerase is associated with radio- and chemoresistance. In the here described study we systematically investigated the influence of telomerase activity (TA) and telomere length on the outcome of radio- and chemotherapy in neuroblastoma. Methods We studied the effects on dominant negative (DN) mutant, wild type (WT) of the telomerase catalytic unit (hTERT) using neuroblastoma cell lines. The cells were irradiated with 60Co and treated with doxorubicin, etoposide, cisplatin and ifosfamide, respectively. Viability was determined by MTS/MTT-test and the GI50 was calculated. Telomere length was measured by southernblot analysis and TA by Trap-Assay. Results Compared to the hTERT expressing cells the dominant negative cells showed increased radiosensitivity with decreased telomere length. Independent of telomere length, telomerase negative cells are significantly more sensitive to irradiation. The effect of TA knock-down or overexpression on chemosensitivity were dependent on TA, the anticancer drug, and the chemosensitivity of the maternal cell line. Conclusions Our results supported the concept of telomerase inhibition as an antiproliferative treatment approach in neuroblastomas. Telomerase inhibition increases the outcome of radiotherapy while in combination with chemotherapy the outcome depends on drug- and cell line and can be additive/synergistic or antagonistic. High telomerase activity is one distinct cancer stem cell feature and the here described cellular constructs in combination with stem cell markers like CD133, Aldehyddehydrogenase-1 (ALDH-1) or Side population (SP) may help to investigate the impact of telomerase activity on cancer stem cell survival under therapy.

Background Increased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies. Methods The basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique. Results Low and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies. Conclusion Progressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.

Proviral DNAs are being measured increasingly as a marker of the efficacy of highly active anti-retroviral therapy (HAART) and is accepted for the early diagnosis of perinatal HIV-1 infections. This requires a standardized test which enables the detection of a wide range of subtypes worldwide including O, N and circulating recombinant forms (CRFs). Based on a previous publication, a PCR – Test for HIV-1 provirus detection in peripheral blood mononuclear cells (PBMCs) was developed. Blood samples from 80 individuals infected with HIV-1 and 20 persons negative for HIV-1&2 from Africa and Germany were tested for the presence of HIV-1 provirus DNA. The primer system used enables the detection of proviral DNA despite the high concentrations of human DNA. The limit of detection was determined to be 5 copies per 105 cells. All 20 samples from persons negative for HIV were negative for HIV-1 proviral DNA while provirus DNA was amplified from 76 of the 80 (95%) samples from persons infected with HIV. The amplified products were detected by gel-electrophoresis, flow cytometry and real-time PCR. All three detection systems provided the same results.

In the present study, the predictive value of ionising radiation (IR)-induced cell death was tested in peripheral blood lymphocytes (PBLs) and their corresponding Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) in an interlaboratory comparison. PBLs and their corresponding LCLs were derived from 15 tumour patients, that were considered clinically radiosensitive based on acute side-effects, and matched controls. Upon coding of the samples, radiosensitivity of the matched pairs was analysed in parallel in three different laboratories by assessing radiation-induced apoptotic and necrotic cell death using annexin V. All participating laboratories detected a dose-dependent increase of apoptosis and necrosis in the individual samples, to a very similar extent. However, comparing the mean values of apoptotic and necrotic levels derived from PBLs of the radiosensitive cohort with the mean values of the control cohort did not reveal a significant difference. Furthermore, within 15 matched pairs, no sample was unambiguously and independently identified by all three participating laboratories to demonstrate in vitro hypersensitivity that matched the clinical hypersensitivity. As has been reported previously, apoptotic and necrotic cell death is barely detectable in immortalised LCL derivatives using low doses of IR. Concomitantly, the differences in apoptosis or necrosis levels found in primary cells of different individuals were not observed in the corresponding LCL derivatives. All participating laboratories concordantly reasoned that, with the methods applied here, IR-induced cell death in PBLs is unsuitable to unequivocally predict the individual clinical radiosensitivity of cancer patients. Furthermore, LCLs do not reflect the physiological properties of the corresponding primary blood lymphocytes with regard to IR-induced cell death. Their value to predict clinical radiosensitivity is thus highly questionable.

A flow cytometric method for identifying, purifying, and expanding endothelial progenitor cells (EPC) derived from peripheral blood is reported. During our experiments, we found that isolation of viable EPC is not possible by using the standard flow cytometric protocols, since erythrocyte lysing influences cell survival. Furthermore, erythrocyte lysing has a high impact on quantative analysis of EPC with 20% lower numbers compared to no-lyse data. The viability of EPCs was tested with a colony forming test after both lysis based FACS of EPCs and without lysing. CD133 and VEGF-R2 revealed as positive markers for EPC selection and 7-amino actinomycin D (7-AAD) to eliminate dead cells. The few purified CD133+ and VEGF-R2+ cells showed strong colony-forming capacity in a human stem cell methylcellulose based medium (colony assay) when isolated by the no-lyse protocol. The colonies showed the typical shape of early EPC-colonies with round immature cells in the centre and dendritic or spindelcell-shaped peripheral cells, which were also immunologically identified as EPC-derived. Compared to this, erythrocyte lysing reagents destroyed even all sorted EPCs. Summarizing the presented data suggest that the use of erythrocyte lysing reagents is neither suitable for cloning nor for counting of endothelial progenitor cells, and no-lyse protocols should be used.

Since preanalytic lysing of erythrocytes remains critical in flow cytometry, we investigated the influence of four lysing procedures on the quantification of leukocyte and CD34+ cells in hematopoietic cell transplants (HCTs). Samples were derived from stem cell–enriched mobilized whole blood collected by apheresis (unselected) and immunologically purified stem cell products (selected) and were measured using the dual-platform (2-PF) method with two flow cytometric systems. Additionally, cells were measured by a volume-based technique (single platform [1-PF]). Results were identical in the 2-PF mode (unselected HCTs, r = 0.998; selected HCTs, r = 0.999). In comparison with the 2-PF results, the single-platform (1-PF) measurements revealed a mean decrease of 59.5% for CD34+ cells (50.8% for CD45+ cells) in unselected HCTs and a mean decrease of 52% for CD34+ cells (49.8% for CD45+ cells) in selected HCTs. In order to check the accuracy of cell quantification using the 1-PF method, leukocyte reference values from hematology counter results were compared with flow cytometric (1-PF)–counted nucleated cells. That analysis revealed good congruency, with r = 0.998 for unselected HCTs and r = 0.999 for selected HCTs. In conclusion, all lysing procedures that we used induced substantial loss of leukocytes and CD34+ cells. As demonstrated by the high accuracy of the 1-PF technique, all erythrocyte lysing procedures caused significant cell loss, which led to inconsistent counting of CD34+ cells in nonvolumetric flow cytometric (2-PF) protocols.

All current-flow cytometric techniques use erythrocyte-lysing procedures before leukocyte analysis. We investigated the impact of four lysing procedures with different flow cytometric techniques on the loss of leukocytes and hematopoietic progenitor cells in blood samples. A total of 280 determinations out of 10 samples were measured by two flow cytometers (FCMs), using a FACS-Calibur (Becton Dickinson) and a particle-analyzing system (PAS) with a "true volumetric unit" (Partec). All samples were prepared with four different commercially available erythrocyte-lysing reagents (n = 10, respectively). CD34(+) cells were determined in relation to counted leukocytes with both FCMs (dual platform determinations, 2-PF). In addition, further immunologic and nuclear staining determinations of cells with and without erythrocyte-lysing procedures were performed in the "true volumetric unit" (single platform mode 1-PF) using the PAS system (n = 10, respectively). In the 2-PF mode, both systems showed identical results for CD34(+) cells (r = 0.997). The comparison of 1-PF and 2-PF modes with immunologic stainings revealed a mean decrease of 34.5% for absolute amounts of CD45(+) cells [in detail: Becton-Dickinson (BD) lysis 40%; Ortho Diagnostics (OD) lysis 31%; Uti lyse (UL) 38%; Cylyse (CL) 29%] and of 41.3% for absolute concentration of CD34(+) cells [in detail: BD lysis 45%; OD lysis 40%; UL lysis 45%; CL lysis 34%] by the lysing procedures. In contrast, the nuclear stainings revealed a mean leukocyte loss of only 5% for the nonlysed samples and of 12% for lysed samples. All investigated lysing procedures induced a large loss of leukocytes and progenitor cells, obviously due to cell membrane destruction as demonstrated for identical samples in the 1-PF and 2-PF modes by immunologic and nuclear staining methods.

The succession in time and space of specific germ cell associations, denoted as spermatogenic stages, is a typical feature of mammalian spermatogenesis. The arrangement of these stages is either single stage (one spermatogenic stage per tubular cross-section) or multistage (more than one spermatogenic stage per tubular cross-section). It has been proposed that the single-stage versus multistage arrangement is related to spermatogenic efficiency and that the multistage arrangement is typical for hominids. In the present work, the arrangement of spermatogenic stages and the spermatogenic efficiency of 17 primate species, comprising Strepsirrhini (Prosimians: Lemuriformes, Lorisiformes), Platyrrhini (New World primates), Catarrhini (Old World primates), and Hominoidea (great apes and humans), were analyzed comparatively by quantitative histological and flow cytometric means. We found a predominant single-stage tubular organization in the Strepsirrhini, indicating that the single-stage form represents the ancestral state. The highest degree of multistage complexity was found in Hominoidea (except orangutan) and in Platyrrhini, but not in Catarrhini. Hence, no direct relationship between single-stage/multistage tubular topography and phylogeny could be established across primates. In fact, the tubule arrangement seen in Platyrrhini and Catarrhini primates is the reverse of what might be expected from phylogeny. Interestingly, spermatogenic efficiency was similar in all species. We found no correlation between single-stage/multistage arrangement and spermatogenic efficiency or mating system. We speculate that the presence of a single-stage/multistage organization might simply reflect germ cell clonal size. Our findings further indicate that sperm competition in primates is not reflected at the level of testicular function.

The quantification of the cellular transport capacity for the DNA staining dye Hoechst 33342 out of the cell is a widely accepted marker for recognition of stem cells. Cells with such an increased level of dye efflux (the so-called side population) can be detected on a flow cytometer equipped with a UV laser. Active transport mechanisms of this type play an important role for the elimination of anti-proliferative agents by cancer cells. A staining method for cells with high dye efflux is decribed.

Higher plants possess two types of glucan phosphorylase (EC 2.4.1.1). One isozyme type, designated as Pho1, is located in the plastid whereas the other type, Pho2, is restricted to the cytosol. For Solanum tuberosum L. two Pho1 type phosphorylases have been sequenced [Nakano, K. & Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236; Sonnewald, U., Basner, A., Greve, B. & Steup, M. (1995) Plant Mol. Biol. 27, 567-576]. Both proteins (referred to as Pho1a and Pho1b, respectively) are highly similar (81-84% amino acid identity over most parts of the two sequences) with the exception of the N-terminal transit peptide and the large insertion located between the N- and the C-terminal domains. In this communication antibodies that bind specifically to either Pho1a or Pho1b were used to study both isoforms at the protein level. The antibodies were applied to both potato tuber and leaf extracts following either denaturing or non-denaturing electrophoresis. Pho1a but not Pho1b was immunochemically detectable in tuber extracts whereas leaf extracts contained both the Pho1a and Pho1b protein. During denaturing electrophoresis the two antigens comigrated. When the leaf Pho1 isoforms were separated by affinity electrophoresis three bands of activity were resolved; all of them were recognized by the anti-Pho1a antibodies, but only two of these reacted with the anti-Pho1b antibodies. The isoform binding exclusively to the anti-Pho1a antibodies comigrated with the Pho1 isozyme from potato tubers. Immunoprecipitation experiments performed with anti-Pho1a antibodies removed the entire Pho1 phosphorylase activity from both tuber and leaf extracts. Addition of anti-Pho1b antibodies to tuber extracts did not affect the enzyme pattern, whereas in leaf extracts one isoform remained unchanged but the two other bands were strongly retarded. This indicates that the Pho1a protein is present in all three forms and Pho1b is associated with Pho1a. Association of Pho1a and Pho1b was further demonstrated by cross-linking experiments using bis(sulfosuccinimidyl)suberate as linker. Immunoprecipitation experiments were also performed using extracts of transformed Escherichia coli cells that expressed either Pho1a or Pho1b or both simultaneously. Under these conditions a homodimeric Pho1b phosphorylase was observed that had a lower electrophoretic mobility than the heterodimer from leaves. In leaves of transgenic potato plants antisense inhibition of the Pho1a gene affected the formation of (Pho1a)2 more strongly than that of the heterodimer. Thus, in leaves, Pho1a exists both as a homodimer, (Pho1a)2 and as heterodimer, (Pho1a-Pho1b); a part of it appears to be covalently modified. Pho1b, in the homodimeric form, is often below the limit of detection. In tubers the homodimer, (Pho1a)2, is the only detectable Pho1-type enzyme. To our knowledge this is the first report on a heterodimeric structure of plant phosphorylase.

Objective To study the function of miR-145, known to be dysregulated in endometriosis, and to identify its target genes in an in vitro endometriosis model. Design Experimental laboratory study. Setting University medical centers. Patient(s) Primary endometrial stroma cells were derived from eutopic endometrium of three American Society for Reproductive Medicine stage III endometriosis patients and from ectopic lesions of four patients with deep infiltrating endometriosis. Intervention(s) The human endometriotic cell line 12Z and primary eutopic and ectopic endometrial stroma cells were transiently transfected with miR-145 precursors or anti–miR-145 inhibitors and investigated for posttranscriptional regulation of predicted target genes and changes in cell behavior. Main Outcome Measure(s) Predicted target expression was measured by quantitative reverse transcription–polymerase chain reaction and Western blotting, and altered cell behavior was monitored by cell proliferation assays. The 12Z cells were additionally investigated by Matrigel invasion assays, cell cycle analysis, side population analysis, and aldehyde dehydrogenase activity assays. Result(s) In all cells investigated, miR-145 overexpression inhibited cell proliferation and induced down-regulation of FASCIN-1, SOX2, and MSI2 . In 12Z cells miR-145 upregulation increased Matrigel invasiveness and reduced side population and aldehyde dehydrogenase-1 activity. Additional down-regulated targets in 12Z cells included OCT4, KLF4, PODXL, JAM-A , and SERPINE1/PAI-1. ACTG2 and TAGLN were up-regulated upon pre–miR-145 transfection. JAM-A, FASCIN-1, and PAI-I down-regulation in 12Z cells were confirmed by Western blotting. Conclusion(s) miR-145 inhibits endometriotic cell proliferation, invasiveness, and stemness by targeting multiple pluripotency factors, cytoskeletal elements, and protease inhibitors.

In potato tubers two starch phosphorylase isozymes, types L and H, have been described and are believed to be responsible for the complete starch breakdown in this tissue. Type L has been localized in amyloplasts, whereas type H is located within the cytosol. In order to investigate whether the same isozymes are also present in potato leaf tissue a cDNA expression library from potato leaves was screened using a monoclonal antibody recognizing both isozyme forms. Besides the already described tuber L-type isozyme a cDNA clone encoding a second L-type isozyme was isolated. The 3171 nucleotide long cDNA clone contains an uninterrupted open reading frame of 2922 nucleotides which encodes a polypeptide of 974 amino acids. Sequence comparison between both L-type isozymes on the amino acid level showed that the polypeptides are highly homologous to each other, reaching 81-84% identity over most parts of the polypeptide. However the regions containing the transit peptide (amino acids 1-81) and the insertion sequence (amino acids 463-570) are highly diverse, reaching identities of only 22.0% and 29.0% respectively. Northern analysis revealed that both forms are differentially expressed. The steady-state mRNA levels of the tuber L-type isozyme accumulates strongly in potato tubers and only weakly in leaf tissues, whereas the mRNA of the leaf L-type isozyme accumulates in both tissues to the same extent. Constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of starch phosphorylase L-type activity in leaf tissue, but had only sparse effects in potato tuber tissues. Determination of the leaf starch content revealed that antisense repression of the starch phosphorylase activity has no significant influence on starch accumulation in leaves of transgenic potato plants. This result indicated that different L-type genes are responsible for the starch phosphorylase activity in different tissues, but the function of the different enzymes remains unclear.

In higher plants several λ-glucan metabolizing enzymes occur as plastid- and cytosol-specific isozymes (1,2). The dual intracellular location of these enzyme activities suggests that both the plastidic and the cytosolic compartment contain a pool of polysaccharides. However, until now a cytosolic starch-like polysaccharide which functions as the physiological carbohydrate substrate of the cytosol-specific isozymes has not been identified and, therefore, the metabolic function of these enzyme forms remains enigmatic.

We demonstrate simultaneously label-free imaging of tumor cell growth and morphology properties as well as the quantification of cellular motion changes that are caused by genetic modifications utilizing digital holographic microscopy.