Elimination of the differential chemoresistance between the murine B-cell lymphoma LY-ar and LY-as cell lines after arsenic (As2O3) exposure via the overexpression of gsto1 (p28).

PURPOSE: Arsenic, in the form of As(2)O(3), has gained therapeutic importance because it has been shown to be very effective clinically in the treatment of acute promyelocytic leukemia (APL). Via numerous pathways arsenic induces cellular alterations such as induction of apoptosis, inhibition of cellular proliferation, stimulation of differentiation, and inhibition of angiogenesis. Responses vary depending on cell type, dose and the form of arsenic. GSTO1, a member of the glutathione S-transferase superfamily omega, has recently been shown to be identical to the rate-limiting enzyme, monomethyl arsenous (MMA(V)) reductase which catalyzes methylarsonate (MMA(V)) to methylarsenous acid (MMA(III)) during arsenic biotransformation. In this study, we investigated whether arsenic trioxide (As(2)O(3)) induces apoptosis in both chemosensitive and chemoresistant cell lines that varied in their expression of p28 (gsto1), the mouse homolog of GSTO1. METHODS: The cytotoxicity of arsenic in the gsto1- and bcl-2-expressing chemoresistant and radioresistant LY-ar mouse lymphoma cell line, was compared with that of the LY-ar's parental cell line, LY-as. LY-as cells are radiosensitive, apoptotically permissive, and do not express gsto1 or bcl-2. Cell survival, glutathione (GSH) levels, mitochondrial membrane potential, and stress-activated kinase status after arsenic treatment were examined in these cell lines. RESULTS: As(2)O(3) induced an equivalent dose- and time-dependent increase in apoptosis in these cell lines. Cellular survival, as measured after a 24-h exposure, was also the same in each cell line. Reduced GSH was modulated in a similar time- and dose-dependent manner. Apoptosis was preceded by loss of mitochondrial membrane potential that triggered caspase-mediated pathways associated with apoptosis. With a prolonged exposure of As(2)O(3), both cell lines showed decreased activation of ERK family members, ERK1, ERK2 and ERK5. As(2)O(3) enhanced the death signals in LY-ar cells through a decrease in GSH, loss of mitochondrial membrane potential, and abatement of survival signals. This effect is similar to that seen when LY-ar cells are treated with thiol-depleting agents or by the removal of methionine and cysteine (GSH precursor) from the growth medium. This response is also completely contrary to that seen for radiation, actinomycin D, VP-16 and other agents, where LY-ar cells do not succumb to apoptosis. CONCLUSIONS: The overexpression of gsto1 in normally chemoresistant and radioresistant LY-ar cells renders them vulnerable to the cytotoxic effects of As(2)O(3), despite the 30-fold overexpression of the survival factor bcl-2. Gsto1 and its human homolog, GSTO1, may serve as a marker for arsenic sensitivity, particularly in cells that are resistant to other chemotherapeutic agents.

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice.

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.

"Mitotic drive" of expanded CTG repeats in myotonic dystrophy type 1 (DM1).

In myotonic dystrophy type 1 (DM1), an expanded CTG repeat shows repeat size instability in somatic and germ line tissues with a strong bias toward further expansion. To investigate the mechanism of this expansion bias, 29 DM1 and six normal lymphoblastoid cell lines (LBCLs) were single-cell cloned from blood cells of 18 DM1 patients and six normal subjects. In all 29 cell lines, the expanded CTG repeat alleles gradually shifted toward further expansion by "step-wise" mutations. Of these 29 cell lines, eight yielded a rapidly proliferating mutant with a gain of large repeat size that became the major allele population, eventually replacing the progenitor allele population. By mixing cell lines with different repeat expansions, we found that cells with larger CTG repeat expansion had a growth advantage over those with smaller expansions in culture. This growth advantage was attributable to increased cell proliferation mediated by Erk1,2 activation, which is negatively regulated by p21(WAF1). This phenomenon, which we designated "mitotic drive" , is a novel mechanism which can explain the expansion bias of DM1 CTG repeat instability at the tissue level, on a basis independent of the DNA-based expansion models. The lifespans of the DM1 LBCLs were significantly shorter than normal cell lines. Thus, we propose a hypothesis that DM1 LBCLs drive themselves to extinction through a process related to increased proliferation.

The nuclear death domain protein p84N5 activates a G2/M cell cycle checkpoint prior to the onset of apoptosis.

In contrast to extracellular signals, the mechanisms utilized to transduce nuclear apoptotic signals are not well understood. Characterizing these mechanisms is important for predicting how tumors will respond to genotoxic radiation or chemotherapy. The retinoblastoma (Rb) tumor suppressor protein can regulate apoptosis triggered by DNA damage through an unknown mechanism. The nuclear death domain-containing protein p84N5 can induce apoptosis that is inhibited by association with Rb. The pattern of caspase and NF-kappaB activation during p84N5-induced apoptosis is similar to p53-independent cellular responses to DNA damage. One hallmark of this response is the activation of a G(2)/M cell cycle checkpoint. In this report, we characterize the effects of p84N5 on the cell cycle. Expression of p84N5 induces changes in cell cycle distribution and kinetics that are consistent with the activation of a G(2)/M cell cycle checkpoint. Like the radiation-induced checkpoint, caffeine blocks p84N5-induced G(2)/M arrest but not subsequent apoptotic cell death. The p84N5-induced checkpoint is functional in ataxia telangiectasia-mutated kinase-deficient cells. We conclude that p84N5 induces an ataxia telangiectasia-mutated kinase (ATM)-independent, caffeine-sensitive G(2)/M cell cycle arrest prior to the onset of apoptosis. This conclusion is consistent with the hypotheses that p84N5 functions in an Rb-regulated cellular response that is similar to that triggered by DNA damage.

Simultaneous estimation of T(G2+M), T(S), and T(pot) using single sample dynamic tumor data from bivariate DNA-thymidine analogue cytometry.

BACKGROUND: Estimating the duration of S phase (T(S) ) and the potential doubling time (T(pot) ) from a single time measurement of the movement of cells using bivariate cytometry is common. However, these estimates require an assumption of the duration of G2 + M (T(G2+M) ). Inspection of the measured dynamic quantities, relative movement [RM(t)], fractions of labeled divided and undivided cells (f(lu)(t) and f(ld)(t)) suggests that T(G2+M), T(S), and T(pot) can be determined simultaneously. METHODS: An equation connecting the growth of the cell population, time, and the dynamic quantities was determined. The equation cannot be solved analytically, but accurate approximations can be used to find T(pot). From this result, the value of T(G2+M) can be determined from f(ld)(t), and T(S) can be determined from RM(t). RESULTS: Kinetic parameters obtained from single time estimates using the new method compared to those obtained from the analysis of multiple time-point measurements of MCa-K and MCa-4 murine tumors are shown to be in close agreement. Moreover, estimates of T(G2+M) in MCa-4 tumors, treated with paclitaxel, provide extra information on the changes in T(G2+M). When applied to the rat R3327-G prostate tumor model following androgen ablation, a correlation analysis of the T(pot) values obtained by the new and previous single time-point methods demonstrates that the rank order from shortest to longest T(pot) values are largely preserved. CONCLUSIONS: The new procedure makes direct estimation of T(G2+M) possible from single time-dynamic measurements. The results from previous studies on T(S) and T(pot) are largely unchanged, but extra information is now available.

Relationship of Ki-67 labeling index to DNA-ploidy, S-phase fraction, and outcome in prostate cancer treated with radiotherapy.

BACKGROUND: Our purpose was to evaluate the relationship of Ki-67 labeling index (Ki67-LI) to deoxyribonucleic acid (DNA) ploidy, S phase fraction (SPF), other clinical prognostic factors, and clinical outcome for patients with prostate cancer treated by external beam radiotherapy. METHODS: Tissue was retrieved from 42 patients who underwent transurethral resection of the prostate before treatment with external beam radiotherapy between 1987-1993. DNA histogram profiles were classified as diploid (diploid + near-diploid) and nondiploid (tetraploid + aneuploid). Immunohistochemical staining of Ki-67 by the MIB-1 monoclonal antibody was used to calculate Ki67-LI. Median patient follow-up was 62 months. Treatment failure was defined as two consecutive rises in serum prostate-specific antigen (PSA) or clinical evidence of disease recurrence. RESULTS: The mean and median Ki67-LIs were 3.1 and 2.4, respectively (range, 0-12.4). Mean Ki67-LI values were significantly associated with higher stage, Gleason score, and pretreatment PSA. Nondiploid tumors had significantly higher Ki67-LIs, as did patients who failed radiotherapy over the follow-up period. SPF was not significantly correlated with Ki67-LI. As a categorical variable, the most significant relationships were seen when Ki67-LI was subdivided into thirds around the median (Ki67-LI 1.5-3.5%, and Ki67-LI >3.5%). This trichotomous variable correlated significantly with pretreatment PSA (P = 0.0008), tumor stage (P = 0.016), Gleason score (P = 0.024), and treatment failure (P = 0.0015), but not with DNA-ploidy (P = 0.15). In actuarial univariate analyses, Ki67-LI appeared to be a more significant predictor of patient outcome (P = 0.003) than DNA-ploidy (P = 0.035). CONCLUSIONS: The Ki67-LI correlated with known prognostic factors such as pretreatment PSA, tumor stage, and Gleason score, and was also weakly related to DNA-ploidy. In comparison to DNA-ploidy, Ki67 LI seems to be a better correlate of treatment outcome.

Enhancement of radiosensitivity of wild-type p53 human glioma cells by adenovirus-mediated delivery of the p53 gene.

OBJECT: The authors sought to determine whether combining p53 gene transfer with radiation therapy would enhance the therapeutic killing of p53 wild-type glioma cells. It has been shown in several reports that adenovirus-mediated delivery of the p53 gene into p53 mutant gliomas results in dramatic apoptosis, but has little effect on gliomas containing wild-type p53 alleles. Therefore, p53 gene therapy alone may not be a clinically effective treatment for gliomas because most gliomas are composed of both p53 mutant and wild-type cell populations. One potential approach to overcome this problem is to exploit the role p53 plays as an important determinant in the cellular response to ionizing radiation. METHODS: In vitro experiments were performed using the glioma cell line U87MG, which contains wild-type p53. Comparisons were made to the glioma cell line U251MG, which contains a mutant p53 allele. Monolayer cultures were infected with an adenovirus containing wild-type p53 (Ad5CMV-p53), a control vector (dl312), or Dulbecco's modified Eagle's medium (DMEM). Two days later, cultures were irradiated and colony-forming efficiency was determined. Transfection with p53 had only a minor effect on the plating efficiency of nonirradiated U87MG cells, reducing the plating efficiency from 0.23 +/- 0.01 in DMEM to 0.22 +/- 0.04 after addition of Ad5CMV-p53. However, p53 transfection significantly enhanced the radiosensitivity of these cells. The dose enhancement factor at a surviving fraction of 0.10 was 1.5, and the surviving fraction at 2 Gy was reduced from 0.61 in untransfected controls to 0.38 in p53-transfected cells. Transfection of the viral vector control (dl312) had no effect on U87MG radiosensitivity. In comparison, transfection of Ad5CMV-p53 into the p53 mutant cell line U251 MG resulted in a significant decrease in the surviving fraction of these cells compared with controls, and no radiosensitization was detected. To determine whether Ad5CMV-p53-mediated radiosensitization of U87MG cells involved an increase in the propensity of these cells to undergo apoptosis, flow cytometric analysis of terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridinetriphosphate nick-end labeling-stained cells was performed. Whereas the amount of radiation-induced apoptosis in uninfected and dl312-infected control cells was relatively small (2.1 +/- 0.05% and 3.7 +/- 0.5%, respectively), the combination of Ad5CMV-p53 infection and radiation treatment significantly increased the apoptotic frequency (18.6 +/- 1.4%). To determine whether infection with Ad5CMV-p53 resulted in increased expression of functional exogenous p53 protein, Western blot analysis of p53 was performed on U87MG cells that were exposed to 9 Gy of radiation 2 days after exposure to Ad5CMV-p53, dl312, or DMEM. Infection with Ad5CMV-p53 alone increased p53 levels compared with DMEM- or dl312-treated cells. Irradiation of AdSCMV-p53-infected cells resulted in a further increase in p53 that reached a maximum at 2 hours postirradiation. To determine whether exogenous p53 provided by Ad5CMV-p53 had transactivating activity, U87MG cells were treated as described earlier and p21 messenger RNA levels were determined. Infection of U87MG cells with Ad5CMV-p53 only resulted in an increase in p21 compared with DMEM- and dl312-treated cells. Irradiation of AdSCMV-p53-infected cells resulted in an additional time-dependent increase in p21 expression. CONCLUSIONS: These data indicate that adenovirus-mediated delivery of p53 may enhance the radioresponse of brain tumor cells containing wild-type p53 and that this radiosensitization may involve converting from a clonogenic to the more sensitive apoptotic form of cell death. Although the mechanism underlying this enhanced apoptotic susceptibility is unknown, the AdSCMV-p53-infected cells have a higher level of p53 protein, which increases further after irradiation, and this exogenous p53 is transcriptionally active. (ABSTRACT TRUNCATE

Resistance of differentiating spermatogonia to radiation-induced apoptosis and loss in p53-deficient mice.

The effect of the p53 gene on the survival of mouse testicular cells was evaluated by analysis of degenerating and terminal transferase-mediated end labeling (TUNEL)-positive cells and the subsequent production of further differentiated progeny. In p53 null mice, in contrast to wild-type mice, radiation induced negligible levels of degenerating or TUNEL-positive differentiating spermatogonia within 24 h. This was correlated with higher production of differentiated progeny of the differentiating spermatogonia in p53 null mice. Contrary to the differentiating spermatogonia, the stem spermatogonia of p53 null mice produced fewer differentiated progeny after irradiation than did the stem cells of wild-type mice. We conclude that, because the degeneration and TUNEL positivity of the differentiating spermatogonia in mice of different genotypes were correlated with each other and were dependent on p53, this process is indeed apoptosis. In the differentiating spermatogonia, p53-dependent apoptosis accounted for the bulk of the loss of their progeny after irradiation. Furthermore, whereas the differentiating spermatogonia died by apoptosis that was dependent on p53, the stem spermatogonia, which are more radioresistant, did not.

Can we detect or predict the presence of occult nodal metastases in patients with squamous carcinoma of the oral tongue?

BACKGROUND: When to do a neck dissection as part of the surgical treatment for a patient with squamous carcinoma of the oral tongue is controversial, particularly when the primary can be resected without entering the neck. If the patient who is at high risk for having occult nodal disease in the neck can be identified, node dissection with the glossectomy could be justified. To better identify patients for this procedure, we correlated various tumor and patient factors along with preoperative diagnostic studies with the presence or absence of pathologically positive nodes in a group of patients who underwent node dissection. METHODS: Ninety-one previously untreated patients with biopsy-proved squamous carcinoma of the oral tongue were prospectively studied. All patients had a glossectomy and neck dissection as their initial treatment. The pathology findings (ie, lymph nodes with squamous cancer) were correlated with many preoperative and intraoperative factors, and a statistical analysis was made. RESULTS: The use of computed tomography and ultrasound was not better than the clinical examination in determining the presence or absence of nodal metastases. The best predictors were depth of muscle invasion, double DNA aneuploidy, and histologic differentiation of the tumor. CONCLUSIONS: All patients with stage T2-T4 squamous cancers of the oral tongue should have an elective dissection of the neck. Patients with T1N0 cancer who have a double DNA-aneuploid tumor, depth of muscle invasion > 4 mm, or have a poorly differentiated cancer should definitely undergo elective neck dissection. Ultrasound and computed tomography are of little value in predicting which patients have positive nodes.

S phase determination in intact colonic crypts by histone H3 messenger RNA in situ hybridization and confocal microscopy.

Proliferating cells have a restricted three-dimensional spatial distribution within the crypt, which is the proliferative unit of the colon. Accurate quantitative and spatial analyses of S phase cells in the colon have therefore been limited by histological techniques. To overcome these limitations, S phase cells in microdissected intact colonic crypts of control, modified-starved, and refed rats were labeled by histone H3 in situ hybridization and analyzed by confocal microscopy. High-resolution digital images of the crypt cell nuclei stained with cyanine nucleic acid and of the labeled S phase cells were produced from confocal microscopic optical crypt sections. The S phase labeling index (LI) per whole crypt significantly (P < 0.001) discriminated the proliferative differences between control, modified-starved, and refed rats and correlated (r = 0.92) with the LI determined from histological crypt sections of the same rats. The variance component of the LI attributable to differences between whole crypts, 0.44 (95% confidence interval, 0.38-0.51), was considerably smaller than that attributable to differences between histological crypt sections, 6.07 (95% confidence interval, 5.18-6.96). Confocal microscopy and histone H3 in situ hybridization of intact three-dimensional crypts enables precise in vitro quantitation and spatial analysis of the total and S phase crypt cells.

Enhanced radioresponse of paclitaxel-sensitive and -resistant tumours in vivo.

Paclitaxel is a potent chemotherapeutic drug and also has the potential to act as a radioenhancing agent. The latter is based on its ability to arrest cells in the radiosensitive G2M phases of the cell cycle; the weight of supporting evidence is derived mainly from in vitro studies. Our previous in vivo experiments identified enhanced tumour radioresponse predominantly attributable to tumour reoxygenation occurring as a result of paclitaxel-induced apoptosis. The current study investigated whether paclitaxel enhanced the radioresponse of tumours which are insensitive to apoptosis induction, but exhibited mitotic arrest, and compared the degree and kinetics of the response to that in tumours which develop apoptosis. The mouse mammary carcinoma MCa-29 (apoptosis sensitive) and the squamous cell carcinoma SCC-VII (apoptosis resistant) were used. In addition, the study investigated whether paclitaxel affected normal skin radioresponse to determine if a therapeutic gain could be achieved. Paclitaxel enhanced the radioresponse of both types of tumours. In the SCC-VII tumour, radiopotentiation occurred within 12 h of paclitaxel administration coincident with mitotic arrest, where enhancement factors (EFs) ranged from 1.15 to 1.37. In MCa-29 tumour, the effect was greater, EFs ranging from 1.59 to 1.91 and occurred between 24 and 72 h after paclitaxel when apoptosis was the predominant microscopic feature of treated tumours and when tumour oxygenation was found to be increased. The acute skin radioresponse and late leg contracture response were essentially unaffected by prior treatment with paclitaxel. Therefore, by two distinct mechanisms, paclitaxel was able to enhance the radioresponse of paclitaxel-sensitive and -resistant tumours, but not the normal tissue radioresponse, thus providing true therapeutic gain.

Supraadditive apoptotic response of R3327-G rat prostate tumors to androgen ablation and radiation.

PURPOSE: Androgen ablation is often combined with radiation in the treatment of patients with prostate cancer, yet, the optimal sequencing and the mechanisms governing the interaction are not understood. The objectives were to determine if cell killing via apoptosis is enhanced when the combined treatment is administered and to define the relationship of changes in this form of cell killing to tumor volume growth delay. MATERIALS AND METHODS: Dunning R3327-G rat prostate tumors, grown in the flanks of Copenhagen rats, were used at a volume of approximately 1 cc. Androgen ablation was initiated by castration, and androgen restoration was achieved with 0.5 cm silastic tube implants containing testosterone. 60Co was used for irradiation. The terminal deoxynucleotidyl transferase (TUNEL) histochemical assay was used to quantify apoptosis. RESULTS: Tumors from intact and castrate unirradiated control rats had average apoptotic indices (percent of apoptotic cells) of 0.4 and 1.0%, respectively. The apoptotic index varied only slightly over time (3 h to 28 days) after castration (range 0.75-1.43%). Irradiation of intact rats to 7 Gy resulted in a peak apoptotic response at 6 h of 2.3%. A supraadditive apoptotic response was seen when castration was initiated 3 days prior to 7 Gy radiation, with peak levels of about 10.1%. When the radiation was administered at increasing times beyond 3 days after castration, the apoptotic response gradually diminished and was back to levels seen in intact rats by 28 days after castration. Tumor volume growth delay studies were consistent with, but not conclusive proof of, a supraadditive effect when the combination was used. DISCUSSION: A supraadditive apoptotic response was seen when androgen ablation and radiation were used to treat androgen sensitive R3327-G rat prostate tumors. This supraadditive effect was dependent on the timing of the two treatments. Further studies are required to more fully define the optimal timing and administration of androgen ablation and radiation.

Quiescence in R3327-G rat prostate tumors after androgen ablation.

Androgen ablation is frequently used in conjunction with radiotherapy in the treatment of high-risk prostate cancer. Androgen ablation-induced cell kinetic changes could result in sub-additive (increased quiescence) or supra-additive (reduction in repopulation) interactions with radiotherapy. The cell kinetic changes were studied in R3327-G Dunning rat prostate tumors grown in vivo using double thymidine analogue labeling and flow cytometry, the terminal deoxynucleotidyl transferase-mediated nick end labeling assay for apoptosis, and measurements of tumor cell numbers. Tumors grown in intact and castrate male rats were continuously labeled for various periods of time with chlorodeoxyuridine and pulse-labeled with iododeoxyuridine 8 h before tumor removal. Androgen ablation resulted in a maximal reduction in labeling index (10 to 1.6%) and an increase in potential doubling time (Tpot; 6-42 days) within 3 days, which was related to a reduction in growth fraction (65% to <10%). In contrast, the length of S-phase was minimally altered (19 to 23 h). The response to androgen ablation involved little apoptosis and no necrosis, and Tpot was approximately the same as the tumor volume doubling time. Hence, the increase in Tpot was mainly the result of a shift to quiescence, and this shift occurred with minimal cell loss. Because quiescence is usually associated with radioresistance, these cell kinetic changes suggest that a sub-additive interaction may occur for some prostate cancers when androgen ablation and irradiation are given together.

The significance of DNA-ploidy and S-phase fraction in node-positive (stage D1) prostate cancer treated with androgen ablation.

BACKGROUND: The prognostic significance of primary tumor DNA-ploidy and S-phase fraction (SPF) was evaluated in patients treated with androgen ablation for regionally localized node-positive prostate cancer. METHODS: All patients were diagnosed with lymph node involvement by pelvic lymphadenectomy between 1984 and 1992 and were treated only with androgen ablation. Median follow-up was 45 months. Adequate material for DNA/nuclear protein flow cytometric analysis was available in 33 patients. RESULTS: The tumors were classified as diploid in 11, near-diploid in 4, tetraploid in 10, and aneuploid in 8 cases. Grouping the patients by nonaneuploidy (diploid and near-diploid and tetraploid) and aneuploidy revealed actuarial 4-year disease progression rates of 14 and 48% (log-rank, P = 0.04), and overall survival rates of 100 and 61% (P = 0.008); however, biochemical progression (rising prostate-specific antigen profile) rates were similar at around 70%. In contrast, SPF was not significantly related to any of the endpoints tested. Several other potential prognostic factors were examined and none correlated significantly with disease progression or survival. CONCLUSIONS: The biochemical progression rates for patients with nonaneuploid and aneuploid tumors were comparable and high, while the disease progression rates were higher and survival rates lower for those with aneuploid tumors. These data indicate that the lead time from biochemical to disease progression and death was shorter with aneuploidy. That these relationships were observed in such a small patient population attest to the strength of DNA-ploidy as a prognostic factor in this cohort.

Protection from procarbazine-induced testicular damage by hormonal pretreatment does not involve arrest of spermatogonial proliferation.

Hormone treatments that suppress sperm production enhance the recovery of spermatogenesis after gonadal exposure to various cytotoxic agents. It has generally been assumed that the mechanism of protection involved an arrest of spermatogonial kinetics. To test this hypothesis critically, we examined spermatogonial kinetics and numbers in rats in which the completion of spermatogenesis was suppressed with a 6-week testosterone plus 17beta-estradiol treatment that protected the testis from procarbazine-induced damage. Histological examination showed that the numbers of A-aligned, intermediate, and B spermatogonia and preleptotene spermatocytes and their mitoses were unaffected by testosterone plus 17beta-estradiol treatment. Flow cytometric analysis of bromodeoxyuridine-labeled cells showed that the percentage of diploid cells undergoing DNA synthesis, the progression of B spermatogonia and preleptotene spermatocytes through S-phase, the division of intermediate and B spermatogonia, the entry of intermediate spermatogonia into their next S-phase as type B cells, and the progression of cells through meiotic prophase were either unchanged or very slightly increased. Thus, changes in spermatogonial numbers or suppression of their proliferation cannot account for protection of spermatogenesis from exposure to cytotoxic agents.

Cell kinetic analysis of intact rat colonic crypts by confocal microscopy and immunofluorescence.

BACKGROUND & AIMS: Precise quantitative and spatial analysis of cell cycle-related biomarkers in colonic crypts is often vital for studies of colon carcinogenesis and cancer prevention. To overcome the limitations of histology, confocal laser microscopy of microdissected whole crypts was used to quantitate S phase and mitotic cells. METHODS: Microdissected distal colonic crypts were studied in a modified rat starvation refeeding model. S phase cells were labeled in vivo with 5-bromodeoxyuridine. Mitotic cells were labeled with MPM2 (antibody to mitosis-specific epitope) and also assessed for chromatin morphology with propidium iodide. Sequential optical crypt sections, produced by confocal microscopy, were digitally imaged. S phase labeling indices per whole crypt were also compared with those derived by conventional immunohistochemistry. RESULTS: S phase and mitotic cells were clearly discriminated without background staining. The labeled S phase cell number and fraction per whole crypt were significantly decreased with starvation and increased with refeeding. Variability in the labeling index between whole crypts analyzed by confocal microscopy was significantly smaller than between histological crypt sections. Consequently, the intervention contributed to 92.2% of the total variability of the labeling index in whole crypts but only to 59% of the variability in histological sections. CONCLUSIONS: Major limitations of histology are overcome by crypt microdissection and confocal microscopic analysis. The total crypt cell population as well as labeled M phase and S phase cells can be imaged, localized, and quantitated with improved precision.

Accelerated repopulation during fractionated irradiation of a murine ovarian carcinoma: downregulation of apoptosis as a possible mechanism.

PURPOSE: To test whether accelerated tumor clonogen repopulation occurs during continuous fractionated radiotherapy of a slow-growing mouse ovarian tumor, and if so whether the accelerated rate of repopulation is predicted by the pretreatment potential doubling time, and whether changes in apoptotic response are a possible mechanism for this change. METHODS AND MATERIALS: The rate of clonogen production during fractionated radiotherapy was followed using the tumor-control assay, with an independent determination of the sensitivity to repeated dose fractions in vivo in the absence of repopulation. The pretreatment potential doubling time was measured by bromodeoxyuridine (BrdUrd) labeling and fluorescence measurements. The apoptotic and mitotic indices at various times during treatment were scored histologically. RESULTS: The slow-growing (pretreatment volume doubling time 6 days) ovarian tumor OCA responds to daily irradiation with 6 Gy under hypoxia by negligible tumor clonogen production in the first few days, followed by a change at about 9 days to accelerated repopulation, after which the effective clonogen doubling time Tclon was about 2 days, near the pretreatment Tpot of 1.7 days. Alternative interpretations of the data, such as a change in radiosensitivity vs. a change in the repopulation rate or acceleration at 3 days as opposed to 9 days, were shown to be unlikely. This change was accompanied by a reduced apoptotic response (measured morphometrically). CONCLUSIONS: When sensitivity to fractionated doses has been corrected for in vivo, this slow-growing mouse tumor exhibits a change to accelerated clonogen production during a continuous radiotherapy regimen that is accompanied or preceded by a reduced histologic apoptotic response. Tclon during accelerated repopulation was slightly longer than the pretreatment Tpot.

Apoptosis and downstaging after preoperative radiotherapy for muscle-invasive bladder cancer.

PURPOSE: To determine the relationship between pretreatment apoptosis levels and clinical-to-pathologic downstaging resulting from preoperative radiotherapy. METHODS AND MATERIALS: Between 1960-1983, 338 patients were dispositioned to receive preoperative radiotherapy 4-6 weeks prior to radical cystectomy for muscle-invasive transitional cell carcinoma of the bladder. Of these, adequate hematoxylin and eosin stained tissue sections for morphologic analysis of apoptosis were available in 158 patients. These patients were treated to a median dose of 50 Gy at 2 Gy per fraction. Median follow-up was 90 months. The apoptotic index (AI) was calculated from the ratio of the number of apoptotic cells divided by the total counted and multiplied by 100. A minimum of 500 cells were counted from each patient. RESULTS: The average AI for the whole group (n = 158) was 2.0 +/- 1.3 (+/- SD), with a median of 1.8. The association of AI to clinical stage was significant with AI averages of 1.8 for Stage T2 (n = 56), 1.9 for T3a (n = 51), and 2.4 for T3b (p = 0.038, Kendall Correlation). The relationship of AI to radiotherapy response also was significant with an average of 2.2 for those who were downstaged (n = 103), 1.9 for those in whom the stage remained unchanged (n = 20), and 1.7 for those who were upstaged (n = 35, p = 0.054, Kendall Correlation). The other significant correlations with AI were for the factors, grade, mitotic index, number of tumors, and gender. The AI was then categorized into three groups ( < or = 1, > 1, and < or = 3, and > 3) to examine the prognostic significance of this parameter. The distributions of patients by clinical stage, grade, mitotic index, number of tumors, radiotherapy response, and hemoglobin level were significantly associated with AI using this grouping. When the analysis of the distribution of patients by radiation response and AI was segregated by stage, a significant correlation was observed only for those with Stage T3b disease (p = 0.006); 93% of T3b patients with an AI > 3 were downstaged, while in 7% the stage remained unchanged and none were upstaged. The relationship of AI to 5-year actuarial patient outcome was investigated using several end points and although no significant correlations were observed, a trend was seen for improved survival when AI was > 3 (71% vs. 41%, p = 0.09) for Stage T3b patients. CONCLUSION: The AI correlated most strongly with radiotherapy response for patients with clinical stage T3b disease, the one subgroup of patients wherein preoperative radiotherapy is likely to be of the most benefit. Further investigation of pretreatment apoptosis levels as a marker of anticancer response is needed, especially for patients treated with chemotherapy and radiotherapy with the goal of bladder preservation.

Histone H3 messenger RNA in situ hybridization correlates with in vivo bromodeoxyuridine labeling of S-phase cells in rat colonic epithelium.

Measurements of cell cycle phase fractions, particularly S-phase, are useful for studies of cell biology and carcinogenesis. Up-regulation of histone gene expression is tightly coupled to the G1-S-phase transition of the cell cycle, and mRNA levels rise 30-100-fold during S-phase. Labeling of histone H3 mRNA using in situ hybridization (ISH) was assessed as a measure of S-phase cells and compared with that found using in vivo 5-bromodeoxyuridine (BrdUrd) labeling in formalin-fixed rat colonic crypts under baseline, modified 72-h starvation, and 24-h refeeding conditions. The labeling index scored in single-labeled sections by histone H3 ISH tightly correlated with that found by in vivo BrdUrd labeling (r = 0.99, p < 0.0001) and clearly discriminated between the control, starved, and refed states (P < 0.001). In 180 crypt sections double labeled using histone H3 ISH and BrdUrd, 92% of 1572 labeled cells exhibited both nuclear BrdUrd and cytoplasmic histone H3 label. It is concluded that histone H3 ISH is an accurate measure of the S-phase fraction and provides an alternative to in vivo BrdUrd labeling in rat colon. This finding warrants validation in human studies.