New Pathways in Cancer Research

UNIVERSIDAD COMPLUTENSE DE MADRID, (Spain)
FACULTAD DE MEDICINA
New Pathways in Cancer Research

RIBOGRAMA: - new relevant biomolecular concept, its
applications in cancer research and other research fields
A. Ferreira-Alemo, MD PhD
The purpose of this research, which was presented as a Doctoral Thesis, at UNIVERSIDAD COMPLUTENSE DE MADRID is draft a method of: - diagnosis, - screening and - monitoring of the colon and rectum cancer, founded on technical and current concepts of Molecular Biology
This new method allows a diagnosis in a "pre-carcinoma in situ stage, since it is based on the phenotype of mucosal cells, composed of very high quantities of free ribosomes in the cytoplasm of colonocytes, which can be quantified by means of the of the flow cytometry, after its isolation, and labelled with fluorochromes.
The repeated records of those quantities of free ribosomes, establishes a graphic curve (RIBOGRAMA) that represents the degree of a malignant tendency, which above a certain level of concentration, allows to say that the cells in a tissue (of the colon and rectum, e.g.) can be considered in the process of developing carcinoma in the colon-rectum mucosa, before any macroscopic viewing (endoscopy).
biomolecular basis of the cellular machinery

FREE AND MEMBRANE RIBOSOMES
ROUGH ENDOPLASMIC RETICULUM
RNA DNA
FREE RIBOSOMES
MEMBRANE RIBOSOMES
CELLULAR NUCLEUS
CYTOPLASM
Free ribosomes are generally assumed to synthesize intracellular proteins (internal economy of the cell), whereas bound ribosomes synthesize proteins intended for secretion (exportation).

MEMBRANE RIBOSOMES
Schematic three-dimensional rough endoplasmic reticulum, showing the synthesis of proteins destined for export by ribosomes attached to membranes reticulum (signal sequence).

CONTROL OF PROTEIN SYNTHESIS
Upstream
side
RIBOSOME
Downstream side
The protein synthetic capacity of a cell is dictated by a number of processes such:

mRNA availability; efficiency of translation; availability of translation factors; number of ribosomes
Tumor Markers

THE RIBOSOME AS THE COMMON ELEMENT IN THE CELLULAR BIOMOLECULAR PROCESS
1. The evidence accumulated to date indicates that overall the protein synthetic capacity is determined by the number of free ribosomes, which function as an informative sensor of the protein synthesis.
2. Increased cell proliferation is a hallmark of agressive malignant neoplasms, which requires a general increase in protein synthesis and a specific increase in the synthesis of replication-promoting proteins.
(Tumor Markers)
3. Ribosome synthesis, or biogenesis, is a complex process dependent on the coordinated synthesis of approximately 85 ribosomal proteins, four ribosomal RNA (rRNA) and their subsequent processing and assembly into mature ribosomes.

THE RIBOSOME AS THE COMMON ELEMENT IN THE CELLULAR BIOMOLECULAR PROCESS
CELL CYCLE
FREE RIBOSOMES QUANTITY
MALIGNANT TRANSFORMED CELL
HYPERPLASIA CELL NORMAL CELL HYPERPLASIA CELL NORMAL CELL
INTERPHASE G1 9,3 S 8,0 Hours G2 2,0
MITOSIS M 0,7 G1 9,3
INTERPHASE S 8,0 Hours

PRO 25,2
MITOSIS G2 2,0 M 0,7
MET 2,1
ANA 2,1
TEL 12,6
Minutes
It has been shown in polyoma virus-transformed cells that one of the key mechanisms for the loss of control of protein synthesis is an inability to decrease ribosome number, which correlated with proliferation of transformed cells in fresh media without addition of serum growth factors (Stanners et al., 1979).
RIBOGRAM CONCEPT
- From the subjective nature, relatively of the amount of free ribosomes, a diagnosis of malignancy is made just under the idea of the constancy property that consists of an great increase of free ribosomes in cells that are in a process of multiplication without self control, as in the case of malignant cells; - A malignant cell has a very high free ribosomes density that is a fundamental aspect of its phenotype; - So, naturally, to describe the quantities of free ribosomes it is necessary to create a language reproducible, not subjective, with mathematical translation, through a graphic curve that we call RIBOGRAMA.
RIBOGRAM CONCEPT
Several lines of evidence, some well established and recent, say emphatically the idea that RNA content is high in cancer cells and genetic events that lead to cancer are often linked directly or indirectly to the ribosome biogenesis.
A perspective on the biology of malignant cells
RIBOGRAM CONCEPT
RIBOGRAMA GRAPHIC CURVE
Legend: smt = every 6 months; A, B, C, D = Concentration levels of free ribosomes
RIBOGRAM CONCEPT
In the decades of 60s and 70s were published studies concerning the accumulation of free ribosomes during chemical induction of neoplastic growth, through initiation by 7,12dimethylbenz(a)anthracene and promotion caused by 12-O-tetradecanoyl-phorbol-13acetate, in the interfollicular areas of the dorsal skin of mice.
Accumulation of ribosomes in the process of oncogenesis

TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION (FIRST 63 CASES AMONG A TOTAL OF 205)
Case Number
Quantitative description of free ribosomes in EM (Electrom Microscope)
Electrom Microscope Main General View Citoplasma filled numerous organelos, mitochondrias, free ribosomes, short
Diagnosis / Information
References (Published in the Doctoral Thesis)
Numerous free ribosomes
Carcinoma pappilar invasivo, moderadamente diferenciado
396
2 3
Free ribosomes randomly dispersed Free ribosomes excedingly abundant
Small clusters of free ribosomes ---------------------
Glial C6 Cells transformadas Human adrenocortical carcinoma derived SW-13 cell lines
397 398
4 5
Numerous free ribosomes Increased free ribosomes
Few granular endoplasmic reticulum Membrane bound ribosomes decreased
Human osteosarcoma Liver tumor rats
399 400
6 7
Free ribosomes Numerous free ribosomes and polysomes
Few organelles Some rough endoplasmatic reticulum ---------------------
Hepatoma cell lines EBV Related liver tumor (occurring after kidney transplant) HEL cells human erytroleukemia induced
401 402
Increase of free ribosome numbers
403
Principal cytoplasmic organelles were free ribosomes into rosettes
Membranes of rough endoplasmic reticulum and Golgi apparatus were poorly developed in most neoplastic cell
Bronchogenic carcinoma
404
10
Rich in ...free ribosomes
Rich in organelles of this type including microtubules mitochondria
Oligodendroglioma
405

Case Number 19
Quantitative description of free ribosomes in EM (Electrom Microscope) exhibited greater activity (free ribosomes)
Electrom Microscope Main General View
Hepatoma transplantable
414
20
Turnover of free ribosomes was greater in host livers
Transplantable hepatoma rat cells
415
21
Free ribosomes were the most prominent Moderate number of mitochondria, were cellular organelles randomly distributed. Poorly developed granular endoplasmatic reticulum
Nasofaringeal carcinoma
416
22
Disagregation of free and membrane bound polyribosomes
----------------------
Effect of aflatoxin B1 on hepatic polyribosomes and protein sintesis in the rat
417
23
Free ribosomes
Few organelles, small number of mitochondria, poor developed Golgi complexes inconspicuous rough endoplasmic reticulum
Keratin positive Ewings sarcoma
418
24
Prominent ultrastructural features being free ribosomes
Prominent - rough endoplasmic reticulum cisternal mitochondria
Human adrenocortical carcinoma derived SW-13 cell line
419
25
Prominent free ribosomes
Prominent rough endoplasmic reticulum
Thalamic tumor in a patient with human T-cell lymphotropic virus type 1 (HTLV-1) [extranodal lymphoma of the skull]
420

Case Number 11
Quantitative description of free ribosomes in EM (Electrom Microscope) Numerous free ribosomes
Electrom Microscope Main General View Enlarged mitochondria prominente smooth and rough endoplasmic reticulum
Mammary Paget Disease
406
12
-----------------------
Intestinal tumor induced by 1,2 dimethylhydrazine
407
13
Increase in membrane free ribosomes
Functional alterations paralleled by an increase in membrane free ribosomes
Rat liver hepatocarcinogenesis induced by 0,25% DL ethiomine [hepatoma] Neuroblastoma cell lines
408
14
cytoplasmicmainly on free ribosomes
Weakly rough surface endoplasmic reticula cisternae and Golgi complexes
409
15
After 2 to 3 weeks of ATRA treatment ER and free ribosomes became rare as the maturation process
Acute promielocytic leukaemia cells
410
16
Cytoplasm may contain free ribosomes
Few mitochondria, a dilated rough endoplasmic reticulum
Metastatic (neck lymph node) malignant extrarenal rhabdoid tumor Ewings sarcoma
411
17
predominantly free ribosomes
Inconspicuous rough -endoplasmic reticulum -small number of mitochondria -developed Golgi complexes
412
18
Free ribosomes were present
Mitochondria endoplasmic reticulum and Golgi apparatus were present
Colorectal polyps
413

Case Number 26
Quantitative description of free ribosomes in EM (Electrom Microscope) Accumulation of numerous free ribosomes
Electrom Microscope Main General View Marcadly increased vesicular trafficking the Golgi
Neuroblastoma B-104 cells
421
27
Free ribosomes
Cytoplasmic organelles such as rough endoplasmic reticulum, mitochondria
Carcinoma of the thyroid gland
422
28
Abundance of dilated rough endoplasmic reticulum
Small cell carcinoma of the ovary
423
29
Abundant free ribosomes
-Absent smooth endoplasmic reticulum
Carcinoma of the endometrium
424
30
Abundant rough endoplasmic reticulum cisternae Smooth endoplasmic reticulum absent
Adrenocortical oncocytoma
425
31
Prominent nucleoli
Chemically induced mouse hepatoblastoma
426
32
Small amounts of rough endoplasmic reticulum and round mitochondria
Malignant fibrous histiocytoma
427
33
Numerous (?) free ribosomes
Numerous small mitochondria
Adenocarcinoma cells of an ovarian clear cell tumor
428

Case Number 34
Quantitative description of free ribosomes in EM (Electrom Microscope) Increase of free ribosomes
Electrom Microscope Main General View Prominent nucleoli, marked decrease in rough endoplasmic reticulum; abundant microphilaments and microtubules
Histogenesis of pseudoductular changes in pancreas of guinea pigs with N-methyl, N-Nitrosourea
429
35
Many free ribosomes
Large nuclei with prominent nucleoli; small quantities of rough and smooth endoplasmic reticulum
NCI H295R cells
430
36
Free ribosomes were common
-Rich mitochondria - Some rough endoplasmic reticulum
Leiomyoblastoma of the uterus
431
37
Increased free ribosomes
Membrane bound ribosomes decreased
Liver tumor rats
432
38 39
High contents of free ribosomes Clusters of free ribosomes
----------------------Dilated endoplasmic reticulum profiles
Sarcoma 45 Cortical dendritic spines
433 434
40 41
Many free ribosomes Numerous free ribosomes
Many rough endoplasmic reticuli -----------------------
Pulmonary clear cell carcinoma Hepatocarcinogenesis induced by ethionine
435 436

TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION (FIRST 63 CASES AMONG A TOTAL OF 205))
Case Number
References
(Published in the Doctoral Thesis)

Human germ cell tumor derived cell line 437
42
...Contain a few cytoplasmic organelles ...except for free ribosomes
Contain few cytoplasmic organelles and a small number of mitochondria
43
tumor cells contains free ribosomes
contain filamentous structures
Embryonal rhabdomyosarcoma in bucal mucosa
438
44
Abundant mitochondria
Oncocytic metaplasia and carcinoma of the endometrium
439
45
referncia a free ribosomes
...Referncia a -Golgi complex -Mithocondria -RER
Tapioca melanoma of the iris...
440
46
free ribosomes were present
Rough endoplasmic reticulum were present
lipomatous lesions in the liver of B6C3F1 mice Pituitary fibrosarcoma following radiotherapy
441
47
numerous free ribosomes
numerous distended rough endoplasmic reticula, Golgi apparatus
442
48
Abundant mitochondria, lysosomes, rough surface endoplasmic reticulum
Epithelioid malignant schwannoma cell line
443

Case Number
References

Rat bladder tumor cell lines 444
49
free ribosomes were observed
desmosomes present
50
increases of free ribosomes
Nuclear lobulation and nucleolar content
Human erythroleukemia cells
445
51
Some mithocondria and other poorly developed cytoplasmic organelles, suggesting undifferentiated nature
Rat malignant fibrous histiocytoma
446
52
Many free ribosomes
-----------------------
Rat colorectal epithelium tumors after treatment with carcinogen
447
53
Poorly differentiated cytoplasm numerous mitochondria
Small cell osteosarcoma
448
54 55
Abundance of free ribosomes Numerous free ribosomes
Paucity of organelles Little rough endoplasmic reticulum; few mitochondria; small Golgi complex
Ewings sarcoma Mouse mammary gland adenocarcinomas
449 450
56
Great number of free ribosomes
-----------------------
Malignant lymphoma
451

Case Number
References

57 Large amount of free ribosomes Well developed Golgi apparatus, rough endoplasmic reticula Monocytic leukaemia cells 452
58
Free ribosomes
Rough endoplasmic reticulum pleomorphic mitochondria
Poorly differentiated Rhabdomyosarcomas
453
59
805 free ribosomes
-----------------------
Lymphocytic leukaemia tumor
454
60
Clusters of free ribosomes
Prominent Golgi apparatus numerous profiles of rough endoplasmic reticulum
Adenocarcinoma from apocrine glands in dogs
455
61
Few free ribosomes
Poor developed Golgi apparatus phenotypic features characteristics for myeloma cells
Myeloma cells in human hybridoma
456
62
Few granular endoplasmic reticulum
Human osteosarcoma
457
63
Many free ribosomes
Numerous organelles many lysosomes, swollen mithocondria, Golgi complexes, rough and smooth endoplasmic reticulum
Subependymal giant cell tumor
458

TABLE II - DIFFERENT WAYS HOW IS DESCRIBED THE CONTENT OF FREE RIBOSOMES IN
ATYPICAL CELLS, ON E.M. - ELECTRON MICROSCOPE (example of the ten cases of table i)
N de caso 1 2 3 4 5 6 7 8 9 10
Descripcin cuantitativa de los ribosomas libres Numerous free ribosomes Free ribosomes randomly dispersed Free ribosomes excedingly abundant Numerous free ribosomes Increased free ribosomes Free ribosomes Numerous free ribosomes and polysomes Increase of free ribosome numbers Principal cytoplasmic organelles were free ribosomes into rosettes Rich in ...free ribosomes
Biomolecular analysis of malignant cells Content of free ribosomes in malignant cells

TABLE III-A - (NUMEROUS FREE RIBOSOMES) Case Number
1 9 10 24 26 30 31 38 44 48
Diagnosis/Information
Papilar Invasive Carcinoma Mammary Paget disease Intestinal Tumor Neuroblastoma Small cell carcinoma of the ovary Malignant fibrous histiocytoma Adenocarcinoma of ovary clear cell Hepatocarcinogenesis induced by ethionine Pituitary fibrosarcoma Rat malignant fibrous histiocytoma
Case Number
50 52 59 75 87 88 91 105 107 108
Small cell osteosarcoma Mouse mammary gland adenocarcinoma Human osteosarcoma Testicular seminoma Phylloides tumor of the prostate Malignant Choroid Plexus papiloma Malignant fibrous histiocytoma Small cell carcinoma of ovary Mammary Paget disease Adenocarcinoma of stomach

TABLE III-A - (NUMEROUS FREE RIBOSOMES) Case Number
117 118
Small cell carcinoma of the ovary Neuroblastoma Rat hepatocarcinogenesis induced by ethionine Small cell osteosarcoma Transplantable Rat malignant fibrous histiocytoma Pituitary adenoma Large malignant choroid plexus Control neurocitoma
Case Number
142 151
Testicular seminoma Mouse mamary gland adenocarcinoma Osteosarcoma Mt TW15 mamosotrofic tumor Hyperplasic foci in precancerous rat liver Human tongue carcinoma cells Intestinal tumors
123 125 127 129 131 132
153 163 178 191 193
LEGEND:
Sample of cases 37 Phenotypes of malignancy observed 35 PERCENTAGE OF MALIGNANT TISSUES - 95%

TABLE III-B - (RICH IN FREE RIBOSOMES)
Case Number
8 96 106 140 197
Diagnosis/ Information
Oligodendroglioma Adenocarcinoma of the stomach Oligodendroglioma Naked nuclei in breast aspirate smears Epidermal carcinogenesis
LEGEND:
Sample of cases 5 Phenotypes of malignancy observed 5 PERCENTAGE OF MALIGNANT TISSUES - 100%

TABLE III-C - (ABUNDANT FREE RIBOSOMES)
Case Number
3 27 28 29 41 45
Case Number
90 95 98 104 115 116
Chemically induced mouse hepatoblastoma Adrenocortical carcinoma Carcinoma of thyroid gland Ductal papillary adenocarcinoma Carcinoma of endometrium Adrenocortical oncocytoma Epithelioid malignant schwannoma cell lise Mouse hepatoblastoma Vaginal hemangiopericytoma Brain tumor
Adrenocortical carcinoma Carcinoma of the endometrium Adrenocortical oncocytoma Chemical induced mouse hepatoblastoma Carcinoma do endometrium Epithelial malignant schwannoma cell line
51 69 71 80
Ewings sarcoma Brain tumor in doges Vaginal hemangiopericytoma Mouse hepatoblastoma
128 138 145 146

TABLE III-C - (ABUNDANT FREE RIBOSOMES) Case Number
156 160 165 166 168 171 173
Extra skeletal Ewings sarcoma Neuroblastoma Epithelial tumors induced in rat kidney Hepatoblastoma of foetal liver Hepatocellular tumors
Case Number
176 183 187 188 189 199 201
Adrenal cortical tumor Pseudolymphoma of the lung Salivary gland carcinoma Epithelioid sarcoma Malignant epithelioid schwannoma Primary lymphosarcoma of testis Ependymoma
Stromal sarcoma of breast Cholangiocarcinoma
LEGEND:
Sample of cases 36 Phenotypes of malignancy observed 34 PERCENTAGE OF MALIGNANT TISSUES - 94,4%

TABLE III-D - (MANY FREE RIBOSOMES)
Case Number
33 37 49 60 73 89 101 102 112 122
NCI-H295 R cells Pulmonary clear cell carcinoma Colorectal epithelium tumors Subependymal giant cell tumor Pancreatic islet cell tumor Neuroblastoma cell line Primary osteoblast like cells Human pituitary tumor NCI-H 295 R cells (human adrenal cell lines) Pluripotent human germ cell tumor derivated cell line
Case Number
124 126 130 137 143 155 164 196 202
Pulmonary clear all carcinoma Colorectal tumors Human neuroblastoma cell line Malignant fibrous histiocytoma Pancreatic islet cell tumor Malignant lymphoma Spontaneous testicular neoplasm Epidermis experimental carcinogenesis Malignant lymphoma
LEGEND:

TABLE III-E - (INCREASED NUMBER OF FREE RIBOSOMES)
Case Number
6 11 15 19 22 23 32
Human erythroleukemia cells Rat liver hepatocarcinoma induced Ewings sarcoma Nasofaringeal carcinoma Adrenocortical carcinoma Thalamic tumor Pseudoductular changes in pancreas pigs
Case Number
62 77 79 84 100 109 110 113 120 133
Papillary thyroid carcinoma Medullary carcinoma of the thyroid Cutaneous neurofibromas R and T head and neck carcinomas Human erythroleukemia cells Transplantable hepatomas Adrenocortical carcinoma Pseudo-ductular changes in pancreas of pigs Embryonal Rhabdomyosarcoma Seminoma and parathyroid adenoma
34 35 47
Leiomyoblastoma of the uteros Liver tumor rats Human erythroleukemia cells

TABLE III-E - (INCREASED NUMBER OF FREE RIBOSOMES)
Case Number
141 157 159 167
Medullary carcinoma Oesophageal epithelium dysplastic foci of the thyroid Pituitary adenomas Uveal malignant melanoma
Case Number
181 185 186 204
Malignant fibrous histiocytoma of the orbit Clear cell thyroid carcinoma Hepatocarcinoma Carcinogenesis induced by 4dimethylaminobenzene
LEGEND:

TABLE III-F - (FREE RIBOSOMES) Case Number
2
Glial C6 cells transformed Administration of ethionine female rats
Case Number
39
Human germ cell tumor derived cell line
5 7 12 14 16 17 21 25 36
40 42 43
Embryonal rhabdomyosarcoma Tapioca melanoma of the iris Lipomatous lesions in the liver of B6 C3 F1 mice
Bronchogenic carcinoma Neuroblastoma cell lines Malignant extra renal rhabdoid tumor Colorectal polyps Hepatoma transplantable Ewings sarcoma Carcinoma of thyroid gland Cortical dendritic spines
46
Rat bladder tumor cell lines Poorly differentiated rhabdomyosarcoma
55 56 57 63 64
Lymphocytic leukaemia tumor Adenocarcinoma from aprocrine glands in dogs Carcinoma of lung Friend erythroleukemia cell growth

65 66 67 68 70 72 74
Hodgkins disease Hepatoma cells Hepatoma cells Astroblastoma Endometrial adenocarcinoma Clear odontogenic tumor Cell condrosarcoma
Case Number
82 83 85 86 92 94 97
Rat pheochromocytoma cell Carcinoma of endometrium Myelogenous leukaemia cell line Seminoma and parathyroid adenoma Myeloma cells Congenital fibrosarcoma Human tumoral ependymal cell lines
76 78 81
Sphenoidol ridge meningioma Osteosarcoma cell line Malignant fibrous histiocytoma
99 103 111
Ductal adenocarcinoma of the pancreas Carcinoma rat testis Human adrenal cell lines

119 120 121 134 135 136
Cerebral small cell tumor Embryonic rhabdomyosarcoma Nevic corpuscle Myelogenous leukaemia cell line Carcinoma of the endometrium Adrenal chromaffin and pheochromocytoma N-methyl-N-amilnitrosamina
Case Number
150 154 158 161 162 169
Carcinoma of the lung Adenocarcinomas (in dogs), aprocrine glands Branchiometric paragangliomes Adrenocortical carcinomas Melanocytomas of the optic nerve Transitional cloagenic carcinomas
139
induced rat oesophageal carcinogenesis
170
Maxilofacial synovial sarcoma
144 148 149
Clear odontogenic tumor Rat ascitis hepatoma cells Rat ascitis hepatoma cells
174 175 177
Cultured hepatoma cells Monoclonal Hodgkin cells cultured Gastric leiomyosarcoma

TABLE III-F - (FREE RIBOSOMES)
Case Number
180 182 184 195 198 200 205
Establishment of a human rectal cancer cell line Pituitary adenomas Myeloma cells Adenocarcinoma of the human tuba uterina Pituitary thyrotrophic tumor Dermatofibroma (histiocytoma) Friend erythroleukemia cell growth
LEGEND:
Sample of cases- 70 Phenotypes of malignancy observed 67 PERCENTAGE OF MALIGNANT TISSUES - 95,7%

TABLE OF "GROUPS OF EXPRESSIONS" RELATIVELY TO DESCRIBE THE CONTENT OF FREE RIBOSOMES
Table of "groups of expressions"

I - (NUMEROUS FREE RIBOSOMES) II (RICH IN FREE RIBOSOMES) III (ABUNDANT OF FREE RIBOSOMES) IV (MANY FREE RIBOSOMES) V (INCREASED NUMBER OF FREE
Number of cases in each group of expressions
Malignant phenotypes in each "group of expressions"
% Malignant tissues
37 5 36 19 38
35 5 34 17 35 67
95 % 100 % 94,4 % 89,4 % 92,2 % 95,7 %
RIBOSOMES) VI (FREE RIBOSOMES) 70

THE RIBOSOME NUMBER AS A QUANTITATIVE PARAMETER OF THE MALIGNANT PHENOTYPE TREND
1.
The RIBOGRAME graphic curve reflects a dynamic idea of changes in numerical values or quantities of all the free ribosomes. The density of free ribosomes is the trend of the phenotype of the malignancy of a cell, relatively to a normal range, of a community of cells under observation in regard to biomolecular behavior, representing a malignant growth of the cell phenotype, above a certain level of increase. It will be very important produce predictive mechanistic models based on tumor dynamics, reflecting the translation in the cell phenotype, for example (the count of free ribosomes) in a cell as the phenotypic alteration process under the oncogenic stimulus.
2.
3.
THEORETICAL CONSIDERATIONS ABOUT THE GRAPH CURVE OF RIBOGRAMA AND ITS IMPLICATIONS IN THE CLINICAL PRACTICE AND OTHER RESEARCH FIELDS
Legend: smt = every 6 months; A, B, C, D = Concentration levels of free ribosomes
INTERPRETATION OF THE GRAPH IC CURVE (RIBOGRAMA) Curve that starts at the normal level and reaching the increased level, which involves care for a prophylactic study on the reasons or causes that determine its appearance. The cells at this level (increased) still have no security features of malignancy, but their increased number of ribosomes continues growing. The cell is transformed into a malignant phenotype (alarm level). At this point it is essential to make a dietary study to survey and identify possible mutations responsible for the uncontrolled growth of ribosome biogenesis in the process of carcinogenesis; Segment of the curve derived from the anterior (segment a-b), which already corresponds to the phenotypic alterations seen in electron microscopy, in which cells are already present with phenotypic features of malignancy (significant increase in median density of free ribosomes), which is the alarm level
Segment of the curve, continuing the anterior segment, which represents the level of signs and symptoms of the CRC disease (clinical level); This segment of the curve corresponds to a change in direction of the curve a-b that results from preventive action and treatment (medications and special diet) Corresponds to a curve in which cells grow (multiply or proliferate) in a self-proliferation mechanisms increased, as in the case of the seminiferous tubules, or the endometrium. This increased level of ribosome biogenesis control is thereof within the physiological (non-malignant); Normal curve This work is intended to study the item colorectal cancer (CRC), in which ribograma curve is high, so it is not the time to talk about the meaning of it at low level. Still, in this level, it will be useful to study conditions of degeneration and involution of cells and tissues, either the intestinal mucosa, or other extraintestinal tissue.

CHARACTERISTICS OF MALIGNANCY VERSUS THE COUNTING OF FREE RIBOSOMES
While Ribograma method gives a quantity idea of the cell phenotipic view, the descriptions of Electron Microscope (EM) view of the cells with phenotypic characteristics of malignancy vary by greater or lesser degree of differentiation of its texture or morphology, but these descriptions are subjective, depending on the observer. In terms of molecular biology of cancer, the malignant transformation of the normal cells is the acquisition of a series of progressive specific genetic changes that act disobeying to the strong antitumor mechanisms that exist in all normal cells, which include: a) - regulation of signal transduction, b) - differentiation, c) - apoptosis, d) - DNA repair; e) - cell cycle progression, f) angiogenesis, g) - cell adhesion, which are not quantifiable, and in its whole do not allow a monitorization reproducible and practical . As in the descriptions of EM, these mechanisms of malignant transformation cannot be evaluated by mathematic criteria, because the described features cannot be quantified. Similarly, the properties of malignant transformed cells, growing in cell culture or in vivo, as observed by techniques of cell biology or molecular biology do not allow a rigorous quantitative assessment, because they depend on subjective criteria, without concrete numbers or standardized numerical limits.
CHARACTERISTICS OF MALIGNANCY VERSUS THE COUNTING OF FREE RIBOSOMES
A.
The characteristics listed in next table are properties of malignant transformed cells growing in cell culture (or in vivo), that are not susceptible of being quantified, while with the quantification free ribosomes it is possible to make comparisons, through counting them with the use of flow cytometry techniques. Then, with the count of free ribosomes is possible to compare the results with reference to the time variable, like it is done with the RIBOGRAMA concept. Of the characteristics listed in the next table it is not possible to register a numeric result, because they do not show a mathematic and reproducible dynamic trend growth and proliferation status of a set of cells belonging to a given tissue, but This is possible with the RIBOGRAMA concept.
B.
C.
PROPERTIES OF GROWING MALIGNANT TRANSFORMED CELLS IN A CULTURE OF CELLS AND /OR IN VIVO
1. Cytological changes similar to cancer cells in vivo, including increased cytoplasmic basophilia, number and size of the nuclei increased, increased nucleuscytoplasm relation, formation of clusters and cords of cells; 2. Altered growth characteristics; a. "Immortality" of the transformed cells in culture. Transformed malignant cells become "immortal" in that they can be transferred in culture indefinitely; b. Decreased dependent inhibition of cell density or loss of "contact inhibition." The transformed cells often grow to a density higher than their normal counterparts, and they can pile up in culture rather than stop growing when they contact each other; c. Decreased serum needs. The transformed cells require decreased concentrations of serum or growth factors to replicate in culture compared to cells not transformed. d. Loss of anchorage dependence and acquisition of the ability to grow in soft agar. The transformed cells may lose their need to grow attached to surfaces and can grow as colonies in semisolid free. e. Loss of cell cycle control. The transformed cells do not stop in G1, or borderline G1 / S cell cycle when subjected to metabolic constraints of growth. f. Resistance to apoptosis (programmed cell death). 3. Changes in the structure and function of the cell membrane - including increased agglutination by plant lectins, altered composition of cell surface glycoproteins, proteoglycans, glycolipids and mucins; appearance of tumor-associated antigens, and increased uptake of amino acids, hexoses and nucleotides. 4. Loss of cell-cell and cell-extracellular matrix that promotes cell differentiation. 5. Loss of response to inducers of differentiation and altered cellular receptors for these agents.
PROPERTIES OF GROWING MALIGNANT TRANSFORMED CELLS IN A CULTURE OF CELLS AND /OR IN VIVO
6. Alteration of signal transduction mechanisms, including growth receptors constitutive phosphorylation cascades and mechanisms of dephosphorylation rather than a regulated function. 7. Increased expression of oncogenic proteins due to translocation, amplification and chromosome mutation. 8. Loss of protein products of tumor suppressor genes due to deletion or mutation. 9. Genomic misreading that causes the overproduction of growth-promoting substances, eg, IGF-2. 10. Increase, or unruly production, of growth factors, e g, TGF-alpha, tumor angiogenesis factor, PDGF, hematopoietic growth factors (e g, CSFs, interleukins). 11. Genetic instability, leading to progressive loss of regulated cell proliferation, enhanced invasion and increased metastatic potential. Genes "mutator" may be involved in this effect. 12. Altered enzyme profifes. The transformed cells have increased levels of enzymes involved in the synthesis of nucleic acids and produce higher levels of lytic enzymes, e g, proteases, collagenases and glycosidases. 13. Production of oncodeveloping gene products. Many malignant transformed cells growing in culture or in vivo produce increased quantities of oncofetal antigen (e g CEA), placental hormones (e g, chorionic gonadotropin), or isoenzymes feto-placental type (e g, placental alkaline phosphatase). 14. Ability to produce experimental animal tumors. This is the condition sine qua non that define the malignant transformation in vitro. If the cells that are thought to have been transformed do not product tumors in appropriate hosts animals, they can not be defined as "malignant". However, the failure to grow in an animal model does not exclude the fact that they can be tumorigenic in a different kind of animal. 15. Ability to avoid host antitumor immune response.
COLORECTAL CANCER
STATISTICS In Europe colorectal cancer (cancer of the colon and rectum) is the most common form of all the cancers. Each year over 200,000 citizens in Europe die from colorectal cancer (nearly two thirds of all cases diagnosed). Colorectal cancer is the third most common cancer worldwide. Yet this disease is preventable in most cases and highly treatable if diagnosed in its very-early stage (pre-in situ stage ).
COLORECTAL CANCER
COLORECTAL CANCER (CRC)
PREPARATION AND COUNTING OF FREE RIBOSOMES
In colorectal cancer, to achieve the quantification of free ribosomes there are, mainly, the following steps:
a) creation of a device (kit to collect stools which have thousands of mucosal cells colonocytes - freed from the colonic and rectal mucosa, which are mixed with and covering the stools voided); b) separation of the cells (colonocytes) from the stools; c) homogenization and calibration of the colonocytes, through rupture of the colonocytes, with certain routine techniques; d) isolation of the free ribosomes, though differential centrifugation; e) labeling of the free ribosomes, utilizing fluorochromes (nanotechnology); and f) counting labeled free ribosomes, utilizing flow cytometry or other method.
COLORECTAL CANCER (CRC) ADVANTAGES OF RIBOGRAMA OVER OTHER NONINVASIVE METHODS OF DETECTECCION OF CRC
The degree of public support will be quite high, given sum of the following nice factors ":
01 02 03 - No invasive act; - No need for the patient, to handle their own feces during collection; - No fecal odor nuisance since the fecal collector is industrially designed accordingly (see the design of the collector of feces in the following images); - No bowel preparation is needed; - No need for cleansing enemas; - It is not necessary to use cathartics; - It is not necessary to shift the patient to a medical appointment or to a medical center;
04 05 06 07
COLORECTAL CANCER (CRC) ADVANTAGES OF RIBOGRAMA OVER OTHER NONINVASIVE METHODS OF DETECTECCION OF CRC
- The patient does not have to change their way of life; - The patient does not have to change the routine of their daily activities or work; 10 - The patient has no need to change the habits and type of food; 11 - The patient does not have to stop, or modify any medication; 12 The method does not give false negatives or false positives, because the test specification is limited to counting the ribosomes of colonocytes, which are the targets to be studied in cases of colorectal cancer, or in cases under malignization process, meaning that the test is 100% specific, 100% reliable and universal, for all tissues, once the cells to study are separated. 08 09
RIBOGRAMA COLO RECTAL CANCER FINAL REMARKS
RIBOGRAMA is an objective and quantitative method that provides an unique service to HealthCare Providers worldwide. The method is qualified to deal within all business sectors of the worldwide HealthCare, from the sole practice physician to the largest chain of hospitals. The advantages of RIBOGRAMA METHOD over other noninvasive tests of cancer of the colon and rectum are the evident twelve "factors nice" that by themselves provide a good patient compliance and a preference for determining the health professionals, because there are no false positives or false negatives, since they are considering charges of ribosomes of cells isolated from the colorectal mucosa.
SUPPORT PROTOTYPE OF STOOL COLLECTION

(COLLECTOR)
SUMMARY OF COLECTOR PARTS

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(COLLECTOR)

(COLLECTOR)

(COLLECTOR)
APLICATIONS
The colorectal cancer represents one of the biggest incidences of human cancer in the western type of life and cocked food. This research project has strong potential in the following fields : public health, medico-hospitalar practice, clinical and laboratorial research, food industry, veterinary, oncologic research, pharmaceutical industry and clinical trials research etc., The method has great medical and financial impact, firstly, by now, in the lowering significantly the morbidity and mortality of the colorectal cancer disease, in the world, and, after, with other organs and systems.
Raise new questions, explore new possibilities, and regard old problems from a new angle... [Albert Einstein]
The place where I was borned and grown. Oporto city - PORTUGAL

New Pathways in Cancer Research

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UNIVERSIDAD COMPLUTENSE DE MADRID, (Spain)

New Pathways in Cancer Research

biomolecular basis of the cellular machinery

ROUGH ENDOPLASMIC RETICULUM

biomolecular basis of the cellular machinery

biomolecular basis of the cellular machinery

The protein synthetic capacity of a cell is dictated by a number of processes such:

biomolecular basis of the cellular machinery

biomolecular basis of the cellular machinery

HYPERPLASIA CELL NORMAL CELL HYPERPLASIA CELL NORMAL CELL

INTERPHASE G1 9,3 S 8,0 Hours G2 2,0

MITOSIS M 0,7 G1 9,3

INTERPHASE S 8,0 Hours

MITOSIS G2 2,0 M 0,7

biomolecular basis of the cellular machinery

biomolecular basis of the cellular machinery

A perspective on the biology of malignant cells

biomolecular basis of the cellular machinery

RIBOGRAMA GRAPHIC CURVE

Legend: smt = every 6 months; A, B, C, D = Concentration levels of free ribosomes

biomolecular basis of the cellular machinery

biomolecular basis of the cellular machinery

Quantitative description of free ribosomes in EM (Electrom Microscope)

References (Published in the Doctoral Thesis)

Numerous free ribosomes

Carcinoma pappilar invasivo, moderadamente diferenciado

Free ribosomes randomly dispersed Free ribosomes excedingly abundant

Small clusters of free ribosomes ---------------------

Numerous free ribosomes Increased free ribosomes

Few granular endoplasmic reticulum Membrane bound ribosomes decreased

Human osteosarcoma Liver tumor rats

Free ribosomes Numerous free ribosomes and polysomes

Few organelles Some rough endoplasmatic reticulum ---------------------

Increase of free ribosome numbers

Principal cytoplasmic organelles were free ribosomes into rosettes

Rich in ...free ribosomes

Rich in organelles of this type including microtubules mitochondria

biomolecular basis of the cellular machinery

Electrom Microscope Main General View

References (Published in the Doctoral Thesis)

Turnover of free ribosomes was greater in host livers

Transplantable hepatoma rat cells

Disagregation of free and membrane bound polyribosomes

Effect of aflatoxin B1 on hepatic polyribosomes and protein sintesis in the rat

Keratin positive Ewings sarcoma

Prominent ultrastructural features being free ribosomes

Prominent - rough endoplasmic reticulum cisternal mitochondria

Human adrenocortical carcinoma derived SW-13 cell line

Prominent free ribosomes

Prominent rough endoplasmic reticulum

biomolecular basis of the cellular machinery

Quantitative description of free ribosomes in EM (Electrom Microscope) Numerous free ribosomes

References (Published in the Doctoral Thesis)

Mammary Paget Disease

Numerous free ribosomes

Intestinal tumor induced by 1,2 dimethylhydrazine

Increase in membrane free ribosomes

Functional alterations paralleled by an increase in membrane free ribosomes

cytoplasmicmainly on free ribosomes

Weakly rough surface endoplasmic reticula cisternae and Golgi complexes

Acute promielocytic leukaemia cells

Cytoplasm may contain free ribosomes

Few mitochondria, a dilated rough endoplasmic reticulum