Flow cytometric analysis of DNA damage and repair in the cells resistant to alkylating agents.

DNA damage in the cells sensitive and resistant to alkylating agents was determined by flow cytometry analysis of cells stained with anti-DNA monoclonal antibody (MOAB) F7-26. MOAB F7-26 interacted with single-stranded regions in alkylated DNA, and the binding of antibody to the cells increased in proportion to the decrease in cell viability. Development of resistance to L-phenylalanine mustard (L-PAM) in A2780 cells was associated with decreased immunoreactivity of DNA with MOAB F7-26. Fluorescence was significantly lower in resistant cells than in sensitive cells, and the difference in the binding of MOAB between two cell types increased with the dose of L-PAM. The enhancement of L-PAM cytotoxicity to resistant cells by buthionine sulfoximine and hyperthermia was accompanied by a proportional increase of MOAB F7-26 binding to DNA. The same relative potential of sensitization regimens was established by cell survival and MOAB staining. The time course of DNA repair established by decrease of MOAB binding after L-PAM removal was similar in sensitive and resistant cells. Resistance of A2780 cells to L-PAM was associated with low initial level of DNA damage and with decreased cytotoxicity per unit of damage. We conclude that resistant cells could be distinguished from sensitive cells by staining with MOAB F7-26 and that the sensitization of resistant cells could be quantitatively predicted by flow cytometry analysis of MOAB binding.

Intercellular transfer of drug resistance.

The effect of L-phenylalanine mustard (L-PAM) on heterogeneous cell populations containing sensitive and resistant cells was evaluated by flow cytometric analysis of DNA damage. Cell cultures were treated with L-PAM for 1 h, fixed, and stained with anti-DNA monoclonal antibody which detects DNA damage induced by alkylating agents. DNA damage was significantly lower in sensitive A2780 cells cocultured with resistant A549 or A2780/PAM cells than in A2780 cells grown separately. Decrease of DNA damage in sensitive cells did not occur when sensitive and resistant cells were grown in common medium without direct contact. Transfer of drug resistance in cocultures was prevented by phorbol ester which is known to inhibit metabolic cooperation via cell junctions. Treatment of cocultures with buthionine sulfoximine increased DNA damage in resistant cells and prevented decrease of DNA damage in sensitive cells. Glutathione (GSH) content in A2780 cells cocultured with A549 cells was significantly higher than GSH content in A2780 cells grown separately. We conclude that decreased response of sensitive cells in cocultures was induced by contact transfer of GSH from GSH-rich resistant cells to sensitive cells. Intercellular transfer of drug resistance demonstrated by analysis of DNA damage was confirmed by colony formation assay. Treatment with L-PAM and Adriamycin killed all cells in A2780/MDR and A549 cultures. Coculture of these lines survived combination treatment because transfer of GSH to multidrug-resistant cells from GSH-rich A549 cells induced resistance to L-PAM and Adriamycin in a single cell. The presence of 2% A549 cells increased resistance of A2780/MDR cells to L-PAM. Phorbol ester eliminated resistance of coculture to combination treatment. Metabolic cooperation between cell subsets with different mechanisms of drug resistance induced resistance to treatment with drugs of different classes (multiclass drug resistance). Inhibition of cell cooperation may improve the response of tumors to combination chemotherapy.

Prognostic indicators including DNA histogram type, receptor content, and staging related to human breast cancer patient survival.

Primary tumors from breast cancer patients were evaluated for the biochemical presence of three steroid cytosolic receptors and by DNA histogram analysis using flow cytometry. These parameters were compared with the histological and staging diagnoses and the patients' survival over a 36-month period. A total of 74 patients with primary breast tumors were evaluated. The breast samples invariably demonstrated a peak population of diploid G0/1 cells which contained 2C amounts of DNA, as determined by mixing experiments using normal human breast tissues or trout erythrocytes as fixed standards. The tumors were classified into five DNA histogram types based on their DNA index distributions established by flow cytometry. These results showed that 21% of the tumors were diploid and indistinguishable from the diploid population of normal breast cells, 8% were hypodiploid, 11% were hypertetraploid, 8% were multiploid, and the remaining 52% were hyperdiploid. The DNA index values varied from 0.78 (hypodiploid) to 2.60 (hypertetraploid). The percentages of S-phase cells were lowest in the diploid and hypertetraploid tumors and highest in the hypodiploid tumors. Among the 24 patients who died during the 36-month follow-up, 92% (22 of 24) were classified in one of the aneuploid groups. Three high-risk groups identified on the basis of survival after 36 months were distinguished: hypodiploid (50% survival); multiploid (43% survival); and hyperdiploid (50% survival). Rates of survival in the diploid and hyperdiploid groups were 87 and 71%, respectively. The hypodiploid group was distinguished by having the lowest mean estrogen cytosolic receptor value [26 +/- 13 (S.D.) fmol/mg], progesterone cytosolic receptor value (13 +/- 15 fmol/ mg), and androgen cytosolic receptor value (less than 1 +/- 1 fmol/mg). In contrast, the diploid tumors had some of the highest receptor values, with mean estrogen cytosolic receptor value equal to 102 +/- 114 fmol/mg, progesterone cytosolic receptor value equal to 74 +/- 110 fmol/mg, and androgen cytosolic receptor value equal to 65 +/- 80 fmol/mg. The lowest survival rates (17% after 36 months) occurred in patients over 67 years of age who had aneuploid tumors, compared to 100% survival in patients over 67 years of age with diploid tumors. Our results demonstrate the value of using flow cytometry and steroid receptor values as supplements to histopathology for the characterization of subgroups of mammary cancer patients. The ability to identify patients with a good prognosis compared to those at high risk of recurrence and death will be valuable in the design of future prospective treatment studies.

Apoptosis in breast carcinomas detected with monoclonal antibody to single-stranded DNA: relation to bcl-2 expression, hormone receptors, and lymph node metastases.

Precise quantitation of apoptotic cells in solid tumors is necessary to determine the role of apoptosis in cancer growth, prognosis, and treatment. In this study, the intensity of apoptotic death was determined in 91 breast carcinomas with a novel cellular marker of apoptosis based on the staining of histological sections with a monoclonal antibody (MAb) to single-stranded DNA. Staining of apoptotic cells with the MAb reflected the decreased thermal stability of DNA induced by the digestion of nuclear proteins, as demonstrated by the elimination of staining in sections reconstituted with histones before heating. The high sensitivity and specificity of apoptosis analysis with the MAb is based on the central role of protease activation in the mechanism and control of apoptosis. Apoptotic indexes (AIs) in breast carcinomas ranged between 0 and 46%. Most of the carcinomas had relatively low AIs, whereas 29 cases were classified as carcinomas with intensive apoptosis (AI >/= 10%). The high level of apoptotic cell death was associated with negative immunostaining for bcl-2 protein, the loss of estrogen and progesterone receptors, high proportion of cells in S-phase, and increased risk of lymph node metastases. There was no correlation between AI and tumor size or p53 immunostaining. Lymph node metastases were detected in 59% of patients with high levels of apoptosis in primary carcinomas and in only 21% of patients with AIs below 10% in primary carcinomas. Thus, the high sensitivity of the MAb assay made it possible to identify a subset of breast carcinomas with intensive apoptosis and markers of poor prognosis. These results demonstrate that the measurement of apoptosis in breast carcinomas provides valuable prognostic information.

Mechanisms and prevention of cancer dissemination: an overview.

Synthesizing data from many experimental and clinical courses, a current working model for tumor dissemination has been constructed. Several aspects of this model are being exploited to therapeutic advantage for the patient with cancer. Surgeons should continue to exercise the principles of en bloc tumor resection and employ maneuvers to minimize iatrogenic tumor dissemination .

Synergistic induction of apoptosis in breast cancer cells by tamoxifen and calmodulin inhibitors.

Breast cancer cells are relatively resistant to the induction of apoptosis (AP) and drug regimens which readily activate apoptotic death, may enhance the antitumor effect. Rapid and intensive induction of apoptosis was observed in estrogen receptor positive and negative breast cancer cell cultures treated with tamoxifen (TMX) combined with the calmodulin antagonists trifluoperazine (TFP) or W7. TMX (1-5 microM) alone or calmodulin antagonists alone did not induce apoptosis. Importantly, intensive apoptosis was also induced by TMX and TFP in the cells obtained from primary human breast carcinomas. Inhibition of the Ca2+ calmodulin signaling pathway is an effective way to activate apoptotic death in epithelial cells. Combination of TMX with non-toxic calmodulin inhibitors may increase the preventive and therapeutic effects of TMX.

Apoptosis and growth-inhibition in sensitive and resistant leukemic-cells treated with anticancer drugs.

To determine the relation between apoptosis and cytotoxicity the number of apoptotic (AP) cells was measured in MOLT-4 and HL-60 cultures treated with adriamycin, etoposide, cisplatin and 1-B-D-arabinofuranosylcytosine (ara-C) in the dose range inducing 20-95% growth inhibition. DNA degradation in individual AP cells was identified with anti-ssDNA monoclonal antibody. There was a near linear relationship between the number of AP cells and drug dose. All drugs induced apoptosis at a comparable level of cytotoxicity. Higher chemosensitivity of MOLT-4 than HL-60 cells was in agreement with the higher number of AP cells. Synergistic cytotoxicity of drug combinations was predicted by apoptosis assay. The number of AP cells was a reliable indicator of chemosensitivity when various cell lines and different drugs were compared. Low doses of cyclohexemide (CHX) inhibited etoposide-induced apoptosis when cells were exposed to both drugs for 6 h. Treatment with CHX alone during longer period or at higher doses induced apoptosis. Inhibition or induction of apoptosis by CHX in the same cell line was determined by the dose of drugs and the time of treatment. MOLT-4/Adr subline developed by continuous exposure to adriamycin was 1.6 - 2.3 fold resistant to adriamycin, etoposide and ara-C by growth inhibition and apoptosis assays. Spontaneous apoptosis induced by medium depletion was decreased in resistant cells. The selection of cells with diminished apoptotic response at early stage of acquired resistance induced pleoitropic drug resistance.

How surgeon age affects post-treatment surveillance strategies for melanoma patients.

The intensity of post-treatment melanoma patient follow-up varies widely among physicians. We investigated whether physician age accounts for the observed variation in surveillance intensity among plastic surgeons. A custom-designed questionnaire was mailed to USA and non-USA surgeons, all of whom were members of the American Society of Plastic and Reconstructive Surgeons. Subjects were asked how they use 14 specific follow-up modalities during years 1-5 and 10 following primary treatment for patients with cutaneous melanoma. Repeated-measures analysis of variance was used to compare practice patterns by TNM stage, year post-surgery, and age. Of the 3,032 questionnaires mailed, 1,142 (38%) were returned. Of those returned, 395 (35%) were evaluable. Non-evaluability was usually due to lack of melanoma patient follow-up in surgeons' practices. Follow-up strategies for most of the 14 modalities were highly correlated across TNM stages and years post-surgery, as expected. The pattern of testing varied significantly by surgeon age for 3 modalities (complete blood count, liver function tests, and chest X-ray), but the variation was quite small. We concluded that the post-treatment surveillance practice patterns of ASPRS members caring for patients with cutaneous melanoma vary only marginally with physician age. Continuing medical education could account for this observation.

Dual immunofluorescent analysis of human peripheral blood lymphocytes.

These studies reveal that a dual immunofluorescent labeling method is useful for enumerating cells from human peripheral blood that bear the helper, suppressor, and/or T-cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate Helper (H) T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for quantitating suppressor (S) T-lymphocytes. FL-conjugated Leu-4 antibodies were used to measure the T-lymphocyte activity. Dual immunofluorescent stained lymphocytes, prepared either from whole blood or by Ficoll-Hypaque, gradient cell separation, were analyzed by flow cytometry. Two light scatter parameters, forward and 90 degree, were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper, suppressor, and T-lymphocyte distributions from 100 controls were as follows: The average percentages +/- SD of the helper and suppressor cells were 41.2 +/- 7.2 and 23.0 +/- 7.2, respectively. The H/S ratio was 1.99 +/- 0.77, while the T-cell distribution on 28 patients was 71.4 +/- 7.7. The Ficoll-Hypaque purified lymphocytes and lysed whole blood lymphocytes compared favorably in their H/S ratios. A comparison was made between the percentages of helper and suppressor cells enumerated by fluorescent microscopy and flow cytometry in which correlation coefficients of 0.80 and 0.86 were determined, respectively. These studies show that helper and suppressor T-lymphocytes can be quantitated simultaneously by flow cytometry, which enables one to correlate the phenotypic activities of two antibodies against cell surface receptors and permits the measurement of a large number of samples in a minimal time.

Melanoma of the trunk: the results of surgical excision and anatomic guidelines for predicting nodal metastasis.

From 1958 through 1969, inclusive, 418 patients with melanoma of the trunk were treated at the M. D. Anderson Hospital and Tumor Institute. Of these, 128 patients (31%) had Stage I disease and were treated by excision with observation of regional nodes in all except five patients. Retrospectively these Stage I patients were analyzed regarding (1) survival, (2) sites and timing of treatment failures, (3) the relation of the primary site to eventual nodal metastasis, and (4) the variables of sex, size, and location on the trunk, which also were correlated with disease control. The results show: (1) actuarial survival rate of 65.7% and 55.7% at 5 and 10 years, respectively; (2) positive regional lymph nodes (RLN) evolved in 34 patients (28%), systemic metastases in 18 patients (15%), local recurrence (LR) in four patients, LR plus RLN in one patient, and intransit metastases in three patients as the first evidence of failure. Over 90% of LR and positive RLN developed within 24 months. Many intransit recurrences and systemic metastases occurred later and account for much of the biologic variability attributed to melanomas: (3) the anatomy of the lymphatics of the trunk as described by Sappey is an excellent guide to the site of first nodal metastasis, (4) a midline or near-midline primary site correlated with regional failure (p less than 0.05). More men failed regionally than did women (p less than 0.05). In retrospective calculation, 184 regional node dissections would have been required for probable salvage of 13 patients (10%) if surgical treatment for subclinical disease had been used routinely.

Apoptosis (programmed cell death) and the evaluation of chemosensitivity in chronic lymphocytic leukemia and lymphoma.

Chronic lymphocytic leukemia and lymphoma cells were treated with antitumor drugs in vitro and analyzed by flow cytometry to measure the number of apoptotic (AP) cells and DNA damage in the cells that escaped apoptotic death. AP cells were identified by a high sensitivity of DNA to thermal denaturation, which induced binding of antibody to single-stranded DNA, and by decreased stainability of cells with the intercalating DNA dye propidium iodide. The appearance of AP cells was prevented by Zn++ and inhibited by phorbol ester. AP cells were induced by alkylating agents, antimetabolites, and anthracyclines. A linear relationship between L-phenylalanine mustard dose and the number of AP cells was observed. A synergistic interaction between drugs was detected by an increased number of AP cells and by the intensity of DNA damage in non-apoptotic cells. A most interesting example of synergism was the combination of alkylating agents with fludarabine. Linearity of dose-response curves, and the capability to detect drug synergism and to evaluate variable response of cells from different patients to single agents and combinations suggest that flow cytometry of apoptosis will provide a basis for chemosensitivity tests in leukemia and lymphoma.

Inhibition of DNA repair in cells treated with a combination of alkylating agents.

Aphidicolin (AP) or hydroxyurea (HU) inhibited DNA repair and enhanced cytotoxicity in human ovarian carcinoma cells A2780 treated with L-phenylalanine mustard (L-PAM) combined with cisplatin or thioTEPA, and in the cells treated with cisplatin combined with thioTEPA. In cultures treated with L-PAM or cisplatin alone post-treatment with AP or HU had no effect on DNA repair and produced only additive cytotoxicity. Post-treatment with AP + HU inhibited DNA repair and enhanced cell killing in cultures treated with L-PAM alone. The inhibitor of protein synthesis cycloheximide protected cells from the cytotoxicity of AP + HU but had no effect on synergistic cell killing produced by DNA repair inhibition. In cisplatin-resistant cells A2780/CP post-treatment with AP + HU enhanced the cytotoxicity of L-PAM, but not of cisplatin. However, in resistant cells treated with cisplatin combined with L-PAM or thioTEPA DNA repair inhibitors decreased IC90 of cisplatin. Treatment of cells with two alkylating agents enhanced the sensitivity to DNA repair inhibitors and eliminated low sensitivity to inhibitors of repair associated with drug resistance.

Apoptosis in human breast and gastrointestinal carcinomas. Detection in histological sections with monoclonal antibody to single-stranded DNA.

We report application of a novel immunohistochemical procedure for the staining of apoptotic (AP) cells in paraffin sections using monoclonal antibody (MAb) to single-stranded DNA. MAb differentiated between apoptosis and necrosis and in contrast to in situ end labelling specifically stained only AP cells. AP carcinoma cells stained with the antibody were detected in 32 of 58 infiltrating human breast carcinomas and in 9 of 15 colon adenocarcinomas. Stromal cells stained with the MAb were observed in all carcinomas, including those in which no AP carcinoma cells were detected. There was a strong positive correlation between the presence of AP cells, loss of hormone receptors and a high proliferation rate in breast carcinomas. AP cells were present in 80-87% of receptor-negative carcinomas, while most of receptor-positive breast carcinomas did not contain AP cells. Apoptosis in tumor cells was detected significantly more frequently among breast carcinomas with high, than among carcinomas with low S-phase fraction. AP cells were present in 93-95% of breast carcinomas which were receptor-negative and had a high S-phase fraction. Immunostaining demonstrated a strong positive correlation between the loss of bcl-2 protein and intensive apoptosis in breast carcinomas. Association between apoptosis and markers of poor prognosis in breast cancer (loss of hormone receptors, intensive proliferation, loss of bcl-2 protein) indicates that apoptotic cell death is typical of more aggressive carcinomas.

Squamous cell carcinoma of the oropharynx: why we fail.

The squamous cancers of the oropharynx in 384 patients were staged according to a T and N system. Three hundred seventy-one of them were treated for cure at a time when modern surgical and radiotherapeutic technics were available. The patient records were analyzed in regard to five year survival, factors influencing treatment failure, and the effect of new primary cancers. Failure to eradicate the cancer at the primary site remains the largest reason for the patients' demise, but as aggressive local and regional treatment becomes more successful, greater numbers of patients survive only to become victims of distant metastases and second primary cancers.

Analysis of survival and disease control in stage I melanoma of the head and neck.

From 1958 through 1969, 357 patients were treated for melanoma of the head and neck. Of these, 166 had invasive, clinical stage I disease. All patients had wide local excision of the primary. Elective regional node dissection was performed in sixty-nine patients and in the remaining ninety-seven observation only was elected. Retrospective analysis of these 166 patients considered (1) survival and disease control, (2) sites and timing of failures, and (3) the effect of sex, site, type of biopsy, skin grafting, and regional node dissection on disease control and survival. More than 80 per cent of the local recurrences developed within the first twenty-four months. Similarly, in the patients not undergoing initial neck dissection, 80 per cent of those who subsequently had clinically positive regional nodes did so within twenty-four months. In the sixty-nine patients undergoing elective regional node dissection, the survival rate was 33.5 per cent at five and ten years in those with histologically positive nodes. Those patients with elective neck dissections having histologically negative nodes had a survival rate of 75.8 and 67.1 per cent at five and ten years, respectively.

Mesenteric arterial infusions of vasopressin for hemorrhage from colonic diverticulosis.

Twenty-four patients with massive rectal hemorrhage and known or subsequently proved colonic diverticular disease had the bleeding site localized by mesenteric angiography and received intra-arterial infusion of vasopressin to arrest the bleeding. In twenty-two patients the bleeding was controlled with the vasopressin infusion whereas in the remaining two, hemorrhage did not stop and surgery was performed. Of the twenty-two patients in whom bleeding was arrested by vasopressin infusion, twelve received no further surgical therapy, five had elective prophylactic surgical resection after a period of hemostasis, and the remaining five underwent segmental resection for bleeding that recurred after cessation of the infusion. Of the twelve patients who were not operated on, three had rebleeding two, four, and twelve months after vasopressin infusion and two of these three patients required surgery. The remaining nine have had no recurrent bleeding for periods ranging from seven to thirty-four months. Of ten patients who had segmental resection after precise localization of the bleeding site and initial control with vasopressin, no one has had recurrent hemorrhage for periods ranging from two to eighteen months.

Cancer metastasis: a product of tumor-host interactions.

Metastasis continues to be the most devastating event for the patient with an established primary cancer. The most significant therapeutic problems are: (1) treatment of patients with established macrometastases, (2) identification of patients who have micrometastases and (3) the development of adequate adjunctive therapies for micrometastases. It is hoped that our evolving understanding of the biology of experimental metastasis and the high level of premium quality laboratory research ongoing in this area will result in further resolution of this clinical problem or, at least, a better understanding of this most extreme expression of the malignant phenotype.

DNA content in human cancer. Application in pathology and clinical medicine.

The relationship between flow cytometry measurements (DNA ploidy, S-phase index) of solid tumors and survival is reviewed. Breast, ovarian, colorectal, pulmonary, urinary bladder, renal, thyroid, and endometrial cervical carcinoma and melanoma are discussed. Correlations between tumor stage or grade and flow cytometry-derived data are considered. Tetraploidy, S-phase indexes, and data derived from paraffin-embedded material have been the basis for seemingly controversial interpretations. Related methods are covered in detail and comparative aspects of flow cytometry and cytophotometry are reviewed.